Reagents for reducing leukocyte interference in immunoassays

ABSTRACT

Methods and devices for reducing interference from leukocytes in an analyte immunoassay are provided. In one embodiment, a method is provided comprising the steps of amending a biological sample such as a whole blood sample with one or more leukocidal reagents that reduce or eliminate the metabolic activity of leukocytes, and performing an immunoassay on the amended sample to determine the concentration of analyte in the sample. Preferably, the sample is amended with one or more enzymes and optionally one or more enzyme substrates and cofactors.

CROSS REFERENCE TO RELATED APPLICATIONS

The present application is a divisional application of U.S. ApplicationNo. 13/862,632, filed on Apr. 15, 2013, now U.S. Pat. No. 9,018,017issued on Apr. 28, 2015, which is a divisional application of U.S.application Ser. No. 12/771,634, filed Apr. 30, 2010, now U.S. Pat. No.8,476,079 issued on Jul. 2, 2013, the entire contents and disclosures ofwhich are incorporated herein by reference.

FIELD OF THE INVENTION

The present invention relates to reducing or eliminating interferencefrom buffy coat components, notably leukocytes, in devices and methodsfor determining the presence or concentration of an analyte in a bloodsample by immunoassay. In particular, the invention relates to reducingor eliminating leukocyte immunosensor interference by amending a bloodsample with a leukocidal reagent that reduces or eliminates the activityof leukocytes.

BACKGROUND OF THE INVENTION

A multitude of laboratory immunoassay tests for analytes of interest areperformed on biological samples for diagnosis, screening, diseasestaging, forensic analysis, pregnancy testing and drug testing, amongother reasons. While a few qualitative tests, such as pregnancy tests,have been reduced to simple kits for a patient's home use, the majorityof quantitative tests still require the expertise of trained techniciansin a laboratory setting using sophisticated instruments. Laboratorytesting increases the cost of analysis and delays the patient's receiptof the results. In many circumstances, this delay can be detrimental tothe patient's condition or prognosis, such as for example the analysisof markers indicating myocardial infarction and heart failure. In theseand similar critical situations, it is advantageous to perform suchanalyses at the point-of-care, accurately, inexpensively and with aminimum of delay.

Many types of immunoassay devices and processes have been described. Forexample, a disposable sensing device for successfully measuring analytesin a sample of blood is disclosed by Lauks in U.S. Pat. No. 5,096,669.Other devices for successfully measuring features such as for exampleclotting time are disclosed by Davis et al. in U.S. Pat. Nos. 5,628,961and 5,447,440. These devices employ a reading apparatus and a cartridgethat fits into the reading apparatus for the purpose of measuringanalyte concentrations and viscosity changes in a sample of blood as afunction of time. The entire contents and disclosures of U.S. Pat. Nos.5,096,669; 5,628,961; and 5,447,440 are incorporated herein by referencein their entireties.

U.S. Pat. Appl. Pub. 2006/0160164 to Miller et al. describes animmunoassay device with an immuno-reference electrode; U.S. Pat. No.7,682,833 to Miller et al. describes an immunoassay device with improvedsample closure; U.S. Pat. Appl. Pub. 2004/0018577 to Emerson Campbell etal. describes a multiple hybrid immunoassay; and U.S. Pat. No. 7,419,821to Davis et al. describes an apparatus and methods for analytemeasurement and immunoassay, each of which is jointly-owned and isincorporated herein by reference in its entirety.

Non-competitive two-site immunoassays, also called sandwich-typeimmunoassays, are often employed for determining analyte concentrationin biological test samples, and are used for example in thepoint-of-care analyte detection system developed by Abbott Point of CareInc., the i-STAT® immunoassay system. In a typical two-siteenzyme-linked immunosorbent assay (ELISA), one antibody is bound to asolid support to form an immobilized or capture antibody and a secondantibody is conjugated or bound to a signal-generating reagent such asan enzyme to form a signal or labeled antibody. Upon reaction with asample containing the analyte to be measured, the analyte becomes“sandwiched” between the immobilized antibody and the signal antibody.After washing away the sample and any non-specifically bound reagents,the amount of signal antibody remaining on the solid support is measuredand should be proportional to the amount of analyte in the sample.

Electrochemical detection, in which the binding of an analyte directlyor indirectly causes a change in the activity of an electroactivespecies adjacent to an electrode, has also been applied to immunoassays.For a review of electrochemical immunoassays, see Laurell et al.,Methods in Enzymology, vol. 73, “Electroimmunoassay”, Academic Press,New York, 339, 340, 346-348 (1981).

In an electrochemical immunosensor, the binding of an analyte to itscognate antibody produces a change in the activity of an electroactivespecies at an electrode that is poised at a suitable electrochemicalpotential to cause oxidation or reduction of the electroactive species.There are many configurations or arrangements for meeting theseconditions. For example, the electroactive species may be attacheddirectly to an analyte, or the antibody may be covalently attached to anenzyme that either produces an electroactive species from anelectroinactive substrate or destroys an electroactive substrate. See,M. J. Green (1987), Philos. Trans. R. Soc. Lond. B. Biol. Sci.,316:135-142, for a review of electrochemical immunosensors.

The concept of differential amperometric measurement is well known inthe electrochemical art. See, for example, jointly-owned U.S. Pat. No.5,112,455 to Cozzette et al., which is herein incorporated by referencein its entirety. A version of a differential amperometric sensorcombination is disclosed in jointly-owned U.S. Pat. No. 5,063,081 toCozzette et al. (the “'081 Patent”), which is herein incorporated byreference in its entirety. The '081 Patent also discloses the use ofpermselective layers for electrochemical sensors and the use offilm-forming latexes for immobilization of bioactive molecules. The useof poly(vinyl alcohol) (PVA) in sensor manufacture is described in U.S.Pat. No. 6,030,827 to Davis et al., which is herein incorporated byreference in its entirety. U.S. Pat. Appl. Pub. 2003/0059954 to Vikholmet al., which is herein incorporated by reference in its entirety,teaches antibodies directly attached to a surface having a biorepellantor biomolecule repellant coating, e.g., PVA, in the gaps between theantibodies on the surface. U.S. Pat. No. 5,656,504 to Johansson et al.teaches a solid phase, e.g., PVA, with antibodies immobilized thereonand is incorporated herein by reference in its entirety, and U.S. Pat.Nos. 6,030,827 and 6,379,883 to Davis et al. teach methods forpatterning PVA layers and are incorporated herein by reference in theirentireties.

It is well known in the art that immunoassays are susceptible to variousforms of interferences. Jointly-owned pending U.S. application Ser. No.12/411,325 (the “'325 Application”), for example, addresses amelioratinginterferences from heterophile antibodies by the inclusion IgM into anIgG reagent cocktail. The '325 application is incorporated herein byreference in its entirety.

As immunoassay technology has increasingly been adapted to enter thepoint-of-care testing market, the use of whole blood as the test mediumhas increased relative to plasma and serum, which are generally used incentral laboratory testing. When whole blood is analyzed, erythrocytesand buffy coat components are present in the assay medium. Those skilledin the art recognize that the buffy coat is a layer of leukocytes andplatelets that forms above the erythrocytes when blood is centrifuged.

It has been found that in certain assays, various assay components,e.g., beads and electrode surfaces, can effectively be opsonized withrespect to leukocytes. For example, with respect to an electrodesurface, Hill et al. (FEBS 191, 257-263, 1985) opsonized amicrovoltammetric electrode with human IgG for the purpose of observingthe respiratory burst of a human neutrophil based on electrochemicaldetection of the superoxide ion.

U.S. Pat. Appl. Pub. 2006/0160164 (the '164 Application), referencedabove, discusses electrochemical immunosensors, the bias betweenwhole-blood and plasma, and provides that immunoassays for markers suchas troponin and the like are generally measured and reported as plasmaor serum values. The '164 Application teaches that when theseimmunosensors are used for analysis of whole-blood, either a correctionfactor or a means for eliminating the bias needs to be employed. The'164 Application further teaches that certain aspects of this bias canbe eliminated, including the bias in whole-blood electrochemicalimmunoassays associated with components of the buffy coat, and also thebias associated with hematocrit variations between samples.

As provided in the '164 Application, leukocyte (or white cell)interference occurs on immunosensors having beads coated with an analyteantibody, e.g., troponin antibody, and control experiments have shownthat this positive bias is absent in plasma samples and in blood sampleswhere the buffy coat has been removed. Thus, it appears that leukocytesare able to stick to the immunosensor and promote non-specific bindingof the enzyme-labeled antibodies, which remain bound even after awashing step. In the '164 Application, it was shown that this bias couldbe partially eliminated by adding a small amount of an antibody to humanserum albumin during bead preparation. Consequently, when a samplecontacts the modified beads, albumin from the sample rapidly coats thebeads and once they are coated with a layer of native albumin theleukocytes should not recognize the beads as an opsonized surface.

The '164 Application describes an additional solution to the leukocyteinterference problem wherein the bias is eliminated by increasing thesalt concentration of the blood sample from a normal sodium ionconcentration of about 140 mM to above about 200 mM, preferably to about230 mM. The mechanism that accounts for reduced interference may be thatthe salt causes osmotic shrinkage of the leukocytes. This interpretationis consistent with the leukocytes' impaired ability to interact with thedisclosed immunosensor.

Notwithstanding the above literature, the need remains for improvedprocesses for ameliorating effects of leukocyte activity in immunoassaysin at least the following areas: immunosensor interference, most notablyin the context of point-of-care testing; electrochemical immunoassays;use of an immunosensor in conjunction with an immuno-reference sensor;whole blood immunoassays; single-use cartridge based immunoassays;non-sequential immunoassays with only a single wash step; and dryreagent coatings.

SUMMARY OF THE INVENTION

The invention is directed to reducing or eliminating leukocyteinterference in kits, devices and methods for using kits and devices,that involve immunoassays. According to various embodiments of theinvention, samples containing an analyte of interest may be amended withone or more leukocidal reagents that reduce or eliminate the activity ofleukocytes, and the resulting amended sample may be analyzed in animmunoassay to determine analyte content and/or concentration withoutsignificant leukocyte interference.

In one embodiment, the invention is to a method of performing animmunoassay for a target analyte in a blood sample, comprisingcontacting a blood sample with a leukocidal reagent, wherein saidreagent substantially inhibits activity of leukocytes in the sample, andperforming an immunoassay on the sample to detect the target analyte. Insome embodiments, the immunoassay is a sandwich assay performed bycontacting the sample with an immunosensor comprising an immobilizedfirst antibody to the target analyte, and a labeled second antibody tothe target analyte. In other embodiments, the immunoassay is acompetitive assay performed by contacting the sample with animmunosensor comprising an immobilized first antibody to the targetanalyte and to a labeled target analyte.

In another embodiment, the invention is to a kit for performing animmunoassay for a target analyte in a whole blood sample. The kitcomprises a leukocidal reagent capable of substantially inhibitingleukocyte activity in the sample, and immunoassay reagents for detectingthe target analyte. In some embodiments, the immunoassay is a sandwichassay and wherein the immunoassay reagents comprise an immunosensorcomprising an immobilized first antibody to the target analyte, and alabeled second antibody to the target analyte. In other embodiments, theimmunoassay is a competitive assay and the immunoassay reagents comprisean immunosensor comprising an immobilized first antibody to the targetanalyte and to a labeled target analyte.

In preferred embodiments, the leukocyte activity that is to be inhibitedis phagocytosis. In certain embodiments of the invention, the leukocidalreagent is an oxygen depleting reagent and/or a glucose depletingreagent. For example, the reagent may comprise glucose oxidase. Inadditional embodiments, the leukocidal reagent comprises glucose oxidaseand glucose. In some embodiments, the leukocidal reagent comprisesglucose oxidase and glucose, and the reagent mixes with the sample toprovide a glucose oxidase activity above about 3 IU per mL and a glucoseconcentration above about 500 mg/dL. In other embodiments, theleukocidal reagent comprises glucose oxidase and an electron acceptor.In still other embodiments, the leukocidal reagent comprises hexokinaseand a source of adenosine-5′-triphosphate (ATP). In other embodiments,the reagent comprises hexokinase, creatine kinase, adenosine diphosphate(ADP), and creatine phosphate. In some embodiments, the leukocidalreagent comprises glucose dehydrogenase and in yet others, the reagentcomprises glucose dehydrogenase, NAD, NADH oxidase, and an electronacceptor. In other embodiments of the invention, the leukocidal reagentis an oxygen depleting reagent. In specific embodiments, the leukocidalreagent is an oxygen depleting reagent selected from the groupconsisting of dithionite, glucose oxidase, and ascorbate oxidase. Inadditional embodiments, the leukocidal reagent is a mitochondrialelectron transport inhibitor and in others, the reagent is amitochondrial membrane uncoupling agent. In other embodiments, theleukocidal reagent is selected from the group consisting of cyanide,antimycin, rotenone, malonate, carbonyl cyanidep-[trifluoromethoxyl]-phenyl-hydrozone, 2,4-dinitrophenol, andoligomycin. In others, the leukocidal reagent is an amphipathic reagent.In still other embodiments, the leukocidal reagent is an amphipathicreagent and is a saponin. In other embodiments, the reagent is aleukocidin and in further embodiments, the reagent is amucopolysaccharide. In certain embodiments, the leukocidal reagent is anAHN-1 antibody and in others, the reagent is lipocortin-1.

In some embodiments, in addition to being contacted with the leukocidalreagent, the sample may be contacted with beads opsonized forleukocytes. For example, in one aspect, the leukocidal reagent comprisesglucose oxidase and glucose, and the sample is contacted with beadsopsonized for leukocytes. In preferred embodiments of the invention, thesample is a whole blood sample. The sample may be amended with ananticoagulant. In some optional embodiments, the analyte is selectedfrom the group consisting of TnI, TnT, BNP, NTproBNP, proBNP, HCG, TSH,NGAL, theophylline, digoxin, and phenytoin.

BRIEF DESCRIPTION OF THE DRAWINGS

These and other objectives, features and advantages of the presentinvention are described in the following detailed description of thespecific embodiments and are illustrated in the following Figures, inwhich:

FIG. 1 is an isometric top view of an immunosensor cartridge cover;

FIG. 2 is an isometric bottom view of an immunosensor cartridge cover;

FIG. 3 is a top view of the layout of a tape gasket for an immunosensorcartridge;

FIG. 4 is an isometric top view of an immunosensor cartridge base;

FIG. 5 is a schematic view of the layout of an immunosensor cartridge;

FIG. 6 is a schematic view of the fluid and air paths within animmunosensor cartridge, including sites for amending fluids with dryreagents;

FIG. 7 illustrates the principle of operation of an electrochemicalimmunosensor with the inclusion of IgM;

FIG. 8 is a side view of the construction of an electrochemicalimmunosensor with antibody-labeled particles not drawn to scale;

FIG. 9 is a top view of the mask design for the conductimetric andimmunosensor electrodes for an immunosensor cartridge;

FIG. 10 illustrates the principle of operation of an electrochemicalimmunosensor with the inclusion of sacrificial beads opsonized forleukocytes;

FIG. 11A shows an unexpected waveform for a brain natriuretic peptide(BNP) cartridge with a positively sloping output signal;

FIG. 11B shows an unexpected waveform for a BNP cartridge with anegatively sloping output signal;

FIG. 11C shows a normal response, which has a near-zero slope for lowanalyte concentrations;

FIG. 11D illustrates that negative slopes are expected only at highanalyte concentrations where the measurement can becomesubstrate-limited rather than enzyme limited;

FIG. 12A shows the effect of oxidative electrode pulsing during the washstep on a normal sample;

FIG. 12B shows the effect of oxidative electrode pulsing during the washstep on an aberrant buffy sample;

FIG. 13 is a schematic illustration of enzymatic regeneration of anelectroactive species;

FIG. 14 illustrates a segment forming means;

FIG. 15 is a top view of the preferred embodiment of an immunosensorcartridge;

FIG. 16 is a schematic view of the fluidics of a preferred embodiment ofan immunosensor cartridge;

FIGS. 17A and 17B show micrographs of immunosensors after assay of BNPin a high buffy sample in the absence (FIG. 17A) and presence (FIG. 17B)of sacrificial beads in the sample;

FIG. 18 illustrates the cartridge device with a slidable sealing elementfor closing the sample entry port in the closed position;

FIG. 19 illustrates the cartridge device with a slidable sealing elementfor closing the sample entry port in the open position;

FIG. 20 illustrate the signal generating reactions occurring during theanalysis: with (a) cleavage of substrate by alkaline phosphatase togenerate electroactive p-aminophenol and (b) oxidation of p-aminophenol;

FIG. 21 shows graphical data for the effect on immunosensor slope inhigh buffy (left panel) and normal whole blood (right panel) sampleswith (sublots 2 and 3) and without (sublot 1) sacrificial microparticlesincorporated in the reagent;

FIGS. 22A and 22B show analyzer waveforms for the associatedimmunosensor micrographs in FIGS. 17A and 17B;

FIG. 23 illustrates the effect of poor washing (inability to replacesample fluid within sensor structure with analysis fluid) onamperometric waveforms;

FIG. 24 illustrates a competitive immunoassay, which may be amended withsacrificial beads according to one embodiment of the invention;

FIG. 25 shows current versus time plots for immunosensors;

FIG. 26 shows the use of glucose oxidase in reducing the effect ofleukocyte activity on the signal output slope of an immunosensor;

FIG. 27 shows a scatter plot for untreated and treated samples and theeffect of leukocyte activity on the signal output slope;

FIG. 28A shows the use of glucose oxidase in reducing the effect ofleukocyte activity on the signal output slope of an immunosensor wherethe sample is retained in a closed container versus open to ambient air;

FIG. 28B shows the use of glucose oxidase in reducing the effect ofleukocyte activity on the signal output slope of an immunosensor wherethe sample is retained in a closed container versus open to ambient air;

FIG. 29 shows the effect of glucose oxidase on simulated arterial bloodsamples; and

FIG. 30 shows the mitigation of phagocytosis in whole blood samples byusing saponin.

DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to the use of leukocidal reagents toreduce or eliminate interference caused by the presence of leukocytes inimmunoassays. In preferred embodiments, the present invention may beemployed in one or more of the following areas: immunosensors, mostnotably in the context of point-of-care testing; electrochemicalimmunoassays; the use of an immunosensor in conjunction with animmuno-reference sensor; whole blood immunoassays; single-use cartridgebased immunoassays; non-sequential immunoassays with only a single washstep; and dry reagent coatings. Notably, while U.S. Pat. Appl. Pub.2006/0160164 (the '164 Application), referenced above, addresses certaininterferences associated with leukocytes based on the addition of ananti-human serum, albumin antibody coating on an immunosensor, and theaddition of salts to the assay medium, the present specificationdiscloses additional sources of bias associated with leukocytes andprovides a novel solution for reducing same. As will be appreciated bythose skilled in the art, the general concept disclosed herein isapplicable to many immunoassay methods and platforms.

The present invention permits rapid in situ determinations of analytesusing a cartridge having an array of analyte sensors and means forsequentially presenting an amended sample to an immunosensor or analytearray. In preferred embodiments, the invention is employed in cartridgesthat are designed to be operated with a reading device, such as thatdisclosed in U.S. Pat. No. 5,096,669 to Lauks et al., issued Mar. 17,1992 (referenced above), or U.S. Pat. No. 7,419,821 to Davis et al.,issued Sep. 2, 2008 (referenced above), both of which are incorporatedby reference herein in their entireties. The invention is bestunderstood in this context. Consequently, a suitable device and methodof operation for a point-of-care immunoassay system is first described,followed by how the system may be best adapted to further reduce oreliminate leukocyte interference in whole blood immunoassays.

In various embodiments, the invention is employed in a heterogeneouselectrochemical immunoassay based on the formation of a sandwich at ornear the electrode surface. In other embodiments, however, the inventionis to other forms of immunoassay. For example, the leukocidal reagentsof the present invention may be employed in either heterogeneous or ahomogeneous bead-based assays, as well as in non-competitive (sandwich)immunoassays or competitive immunoassays. In this context, the terms“heterogeneous” and “homogeneous” refer to the capture step. Hence, forhomogeneous assays, the capture step occurs in the fluid medium, whilein heterogeneous assays, the capture step occurs on a macroscopicsurface, e.g., sensor surface (in either a competitive ornon-competitive manner). In each of these examples, the immobilizedassay beads will be susceptible to attack by leukocytes when the assayis performed in a blood sample. In competitive and non-competitiveassays, this effect can be reduced by the addition of leukocidalreagents and optionally sacrificial beads opsonized for leukocytes.

In a heterogeneous competitive assay, illustrated in FIG. 24, an(unlabeled) analyte present in a sample competes with a labeled analytefor binding sites on a solid surface, e.g., a sensor element. A samplesuspected of containing the analyte of interest (sample analyte) isamended with a quantity of a reagent comprised of the same analyteconjugated to a label, i.e., the labeled analyte. The label may be a dyeor any signal-generating element, such as an enzyme, e.g., ALP. In someexemplary embodiments, the labeled analyte may be labeled with a labelselected from the group consisting of a radiolabel, an enzyme, achromophore, a flurophore, a chemiluminescent species, an ionophore andan electroactive species. In another embodiment, the labeled analyte islabeled with a label selected from the group consisting of afluorescein, a ferrocene (optionally a carboxylated ferrocene ordicarboxylated ferrocene or aminoferrocene), and a p-aminophenol.

Analytes commonly identified and/or measured by means of competitiveassay include the therapeutic agents such as, for example, digoxin,theophylline and biomarkers such as for example C-reactive protein (CRP)as well as phenobarbital, phenytoin, valproic acid and vancomycin. Inpreferred aspects, the sample analyte is selected from the groupconsisting of digoxin, phenobarbital, phenytoin, theophylline, valproicacid and vancomycin. In one embodiment of the invention, the amendedsample is brought into contact with a solid surface on which isimmobilized an antibody to the analyte of interest. The sample-borneanalyte and the labeled analyte compete for binding at this solidsurface so that the amount of labeled analyte and hence the amount ofsignal generated therefrom will be inversely proportional to theconcentration of analyte in the original sample. After washingnon-specifically bound material from the sensor surface, the amount oflabel is measured; in the case of an enzyme label, this would involvesupplying a suitable substrate to the enzyme and detecting the product,e.g., electrochemically or optically. When employed in a heterogeneouscompetitive immunoassay, the blood sample preferably is amended with aleukocidal reagent prior to contacting the solid surface, i.e., sensor.If desired, the blood sample may be further amended with sacrificialbeads opsonized to leukocytes (e.g., IgG-coated microparticles) prior tocontacting the solid surface, as described in U.S. patent applicationSer. Nos. 12/620,179 and 12/620,230, both filed Nov. 17, 2009, theentireties of which are incorporated herein by reference.

I. Cartridge

In one embodiment, the invention relates to cartridges and methods forprocessing liquid samples to determine the presence or amount of ananalyte in the sample. The cartridges preferably contain a meteringmeans, which permits an unmetered volume of sample to be introduced,from which a metered amount is processed by the cartridge and itsassociated reading apparatus. Thus, the physician or operator isrelieved of manually measuring the volume of the sample prior tomeasurement thereby saving time, effort, and increasing accuracy andreproducibility. The metering means, in one embodiment, comprises anelongated sample chamber bounded by a capillary stop and having alongits length an air entry point. Air pressure exerted at the air entrypoint drives a metered volume of the sample past the capillary stop. Themetered volume is predetermined by the volume of the sample chamberbetween the air entry point and the capillary stop.

The cartridge may have a closure device for sealing the sample port inan air-tight manner. This closure device is preferably slidable withrespect to the body of the cartridge and may provide a shearing actionthat displaces excess sample located in the region of the port, therebyreliably sealing a portion of the sample in the holding chamber betweenthe entry port and the capillary stop. See, for example, U.S. Pat. No.7,682,833, the entirety of which is incorporated herein by reference. Inone embodiment, the cartridge may be sealed, for example, by slidablymoving a sealing element over the surface of the cartridge in a mannerthat displaces excess fluid sample away from the sample orifice, seals avolume of the fluid sample within the internal fluid sample holdingchamber, and inhibits fluid sample from prematurely breaking through theinternal capillary stop. The seal obtained by this slidable closuredevice is preferably irreversible and prevents excess blood from beingtrapped in the cartridge because the closure device moves in the planeof the orifice through which blood enters the cartridge and provides ashearing action that seals blood below the plane of the entry port,thereby moving excess blood, i.e., blood above the plane of the orifice,away from the entry port and optionally to a waste chamber.

An exemplary closure device of one embodiment of the present inventionis shown in FIG. 1 and comprises integrated elements 2, 3, 4 and 9 ofcover 1. In this embodiment, closure device 2 rotates about a hingeuntil hook 3 snaps shut blocking sample entry port 4. An alternative tothe closure device comprising integrated elements 2, 3, 4 and 9 of cover1 in FIG. 1 is shown as a separate slidable element 200 in FIGS. 18 and19. FIGS. 18 and 19 show a cartridge device comprising a modifiedversion of the cover of FIG. 1 attached to a base similar to the base inFIG. 4 with intervening adhesive layer 21 shown in FIG. 3 along with theseparate slidable closure element 200. FIG. 19 shows the closure device200 in the open position, where the sample entry port 4 can receive asample, e.g., blood. FIG. 18 shows the closure device 200 in the closedposition where it seals the sample entry port in an air-tight manner. Inoperation, element 200 is manually actuated from the open to the closedposition after the sample, e.g., blood, has been added to the entry portand it enters the holding chamber 34. In the embodiment shown, anyexcess blood in the region of the entry port is moved into an overflowchamber 201 or an adjacent retaining region or cavity. This chamber orregion may include a fluid-absorbing pad or material to retain theexcess sample, e.g., blood.

The sample entry port 4 may be an orifice that is circular, as shown inFIG. 19, or oval and the diameter of the orifice is generally in therange 0.2-5 mm, preferably 1-2 mm, or having a perimeter of 1-15 mm foran oval. The region around the orifice may be selected to be hydrophobicor hydrophilic to control the drop-shape of the applied sample topromote entry into the entry port. One advantage of the closure deviceshown in FIGS. 18 and 19 is that it prevents the sample from beingpushed beyond the capillary stop element 25 at the end of the holdingchamber 34. The presence of a small amount of sample, e.g., blood,beyond the capillary stop is not significant for tests that measure bulkconcentration of an analyte and thus do not depend on sample volume.However, for immunoassay applications where metering of the sample isgenerally advantageous the sealing element improves metering accuracy ofthe device and assures the assayed segment of sample is appropriatelypositioned with respect to the immunosensor when the analyzer actuatesthe sample within the cartridge conduits.

In operation, when the sample, e.g., blood, is added to the cartridge itmoves to the capillary stop. Thus, sufficient sample for the assay ispresent when the region from the capillary stop to the sample entryport, i.e., the holding chamber 34, contains the sample. During theprocess of filling the holding chamber, some sample may remain above theplane of the orifice of the entry port. When the sealing element ismoved from the opened to closed position, any sample that is above theentry port is sheared away without trapping additional sample in the actof closure, thereby ensuring that the sample does not move beyondcapillary stop 25. In a preferred embodiment, sealing element 200 ispositioned within a few thousandths of an inch above the surface of thetape gasket 21 of FIG. 3. The entry port is sealed by the subsequentlowering of the surface of 200 to the adhesive tape gasket when itengages locking features 212 and 213. As the tape is essentiallyincompressible and the orifice has a small diameter, any inadvertentpressure applied to the sealing element by the user will not cause thesample to move beyond the capillary stop.

In certain cartridge embodiments that use several drops of sample, it isdesirable that no bubbles form in the holding chamber as this can affectthe assay. Accordingly, a reliable means for introducing more than onedrop of sample, e.g., blood, into the holding chamber 34 withoutentraining bubbles has been developed. In accordance with one embodimentof the invention, the sample entry port can be designed to receivemultiple drops of sample without successive drops causing trappedbubbles to form in the holding chamber 34 by first treating the holdingchamber with a Corona and/or a reagent cocktail.

The use of Corona treatments on disposable medical devices is well knownin the art and is an effective way to increase the surface activity ofvirtually any material, e.g., metallized surfaces, foils, paper,paperboard stock, or plastics such as polyethylene, polypropylene,nylon, vinyl, polyvinyl chloride (PVC), and polyethylene terephthalate(PET). The Corona treatment makes the materials more receptive to inks,coatings, and adhesives. In practice, the material being treated isexposed to an electrical discharge, or “corona.” Oxygen molecules in thedischarge area break into atoms and bond to molecules in the materialbeing treated, resulting in a chemically activated surface. Suitableequipment for corona treatments is commercially available (e.g., CorotecCorporation, Farmington, Conn., USA). The process variables are wellknown and include the amount of power required to treat the material,the material speed, the width, the number of sides to be treated, andthe responsiveness of a particular material to corona treatment, whichvariables can be determined by a skilled operator. The typical place toinstall a corona treatment is in-line with the printing, coating, orlaminating process. Another common installation is directly on a blownfilm or cast film extruder, because fresh material is more receptive tocorona treatment.

II. Signal Generating Reactions

As described above, the non-competitive (sandwich) immunoassay format isthe most widely used immunoassay method and it is also a preferredformat in the analysis device, e.g., cartridge, discussed herein. Inthis embodiment, one antibody (the immobilized antibody) is bound to asolid support or immunosensor, and a second antibody (the signalantibody) is conjugated/bound to a signal-generating reagent such as anenzyme, e.g., alkaline phosphatase.

Briefly, FIG. 20 illustrates the signal generating reactions occurringduring the analysis with (a) showing the cleavage of substrate byalkaline phosphatase to generate electroactive p-aminophenol and (b)showing oxidation of p-aminophenol. The reaction in (a) occurs in theupper layer of the sensor structure while that in (b) occurs at the goldelectrode surface. The inset in (a) illustrates the pH-dependence of thealkaline phosphatase catalyzed hydrolysis. The central illustrationdepicts the gross features of the immunosensor structure prior toexposure to sample.

The signal-generating reagent (e.g., signal antibody for non-competitiveassays or labeled analyte for competitive assays) may be part of a dryreagent coating in the analysis device, as described below, andpreferably dissolves into the biological sample before the samplereaches the immunosensor. For non-competitive assays, after washing awaythe sample and non-specifically bound reagents, the amount ofsignal-generating reagent (e.g., signal antibody) remaining on the solidsupport should in principle be proportional to the amount of analyte inthe sample. For competitive assays, as discussed above, after washingaway the sample and non-specifically bound reagents, the amount ofsignal-generating reagent (e.g., labeled analyte) remaining on the solidsupport should in principle be inversely proportional to the amount ofanalyte in the sample. However, one limitation of these assayconfigurations is the susceptibility to interference(s) caused byleukocytes present in the blood sample.

III. Leukocidal Reagents

While leukocyte interferences have been mitigated by the means disclosedin jointly-owned U.S. Pat. Appl. Pub. 2006/0160164 to Miller et al.,referenced above, it has now been discovered that for certain bloodsamples, leukocidal activity in a sample, e.g., blood sample, may besurprisingly and unexpectedly reduced or eliminated by the addition ofone or more leukocidal reagents. Thus, in contrast with conventionalprocesses for mitigating leukocyte interference, in various embodimentsof the invention, leukocyte activity may be substantially reduced oreliminated with a leukocidal reagent, defined herein as any compositionthat is capable of metabolically or amphipathically reducing oreliminating leukocyte activity. As discussed below, a non-limiting listof examples of leukocidal reagents includes enzymes and theirco-reagents, saponins, cytotoxins, mucopolysaccharides, leukocidins andanti-leukocyte antibodies. In many embodiments, the leukocidal reagentis an organic, optionally non-ionic, species, while in others thereagent may be inorganic and ionic (e.g., cyanide, dicyanate-containingreagents or dithionite species).

Without being bound by theory, the process of phagocytosis involves anoxidative burst by the leukocytes to generate oxygen radical species,such as superoxide and hydroxyl radicals, that attack a xenobioticentity, e.g., bacteria. As phagocytosis is an oxygen-dependent process,reducing the bulk oxygen concentration in the sample can be used as ameans to prevent or ameliorate leukocyte interference in an immunoassay.Likewise, impeding the metabolic path of these cells in other ways isalso a useful approach to reducing leukocyte interference. In otherembodiments, as described below, sacrificial beads may be used incombination with the leukocidal reagents to reduce or eliminateleukocidal activity.

Several approaches are possible for the metabolic inhibition ofleukocytes. In one embodiment, the leukocytes are deprived of oxygenthat is available in plasma, for example, by using an added enzyme suchas glucose oxidase to consume the glucose and make hydrogen peroxide.This deprives the cells of oxygen, which is the terminal electronacceptor in the respiratory chain. Note that any peroxide in the samplewill quickly be dismutated by catalase to oxygen and water, but the netdriving force of the reaction substantially removes all the oxygen fromthe sample.

In another embodiment, substantially all of the glucose is removed fromthe plasma fraction of the sample. In general, the bulk oxygenconcentration in the blood sample will generally be much lower than theglucose concentration ([O₂]˜100 μM; [glucose]˜5-10 mM). Thus, theaddition of a soluble electron acceptor for glucose oxidase such as, forexample, ferrocenium monocarboxylic acid, phenazine methosulphate, orN,N,N′,N′-tertramethyl-p-phenylene diamine, is desirable for removingsubstantially all the glucose. Alternatively, a glucose dehydrogenase(GDH) can be used; however addition of the cofactor NAD+ is required,with the exception of a GDH with the PQQ cofactor. In this example NADHoxidase (diaphorase) can also be added along with an electron acceptor,as indicated above. In another embodiment, hexokinase may be added,which phosphorylates the available glucose in the sample. Here, a sourceof ATP is required, which is preferably made in situ by adding creatinephosphate, ADP and creatine kinase. In this example only the bulkglucose concentration is affected and not the oxygen concentration.

Generally, it has been found that it is more effective to removesubstantially all of the oxygen rather than substantially all of theglucose in the plasma fraction of the sample. This is related to thefact that the cells already have stored energy (reducing equivalent),but substantially lack an “oxygen store.” Particular embodiments aredirected to the elimination of oxygen though a chemical means, forexample, by the addition of an excess of dithionite, or enzymatically bythe addition of ascorbate oxidase, which reduces oxygen directly towater.

In some embodiments, the invention is directed to methods of performingan immunoassay for a target analyte in a blood sample. In one aspect,the method comprises the step of contacting a sample, e.g., a bloodsample, with a leukocidal reagent, wherein the reagent substantiallyinhibits activity of leukocytes in the sample, and performing animmunoassay on the sample to detect a target analyte. In certainembodiments of the invention, the reagent is an oxygen depleting regentor a glucose depleting reagent or both. In one aspect, the reagentcomprises glucose oxidase. In other aspects, the reagent comprisesglucose oxidase and glucose. In other embodiments, the leukocidalreagent comprises glucose oxidase and glucose, and the leukocidalreagent mixes with the sample to provide a glucose oxidase activityabove about 3 IU per mL and a glucose concentration above about 500mg/dL.

As indicated above, another approach to reducing leukocyte interferenceis to use an electron transport inhibitor or uncoupling agents thatinhibit the mitochondria within the leukocytes. Thus, in one aspect, theleukocidal reagent comprises an electron transport inhibitor. Suitableelectron transport inhibitors and uncoupling agents include, but are notlimited to, rotenone, antimycin, cyanide, malonate (a succinatedehydrogenase inhibitor), 2,4-dinitrophenol (DNP), carbonyl cyanidep-[trifluoromethoxy]-phenyl-hydrazone (FCCP) and oligomycin (aninhibitor of oxidative phosphorylation). While malonate and cyanide canbe added directly to the sample (for example in reagent coating in animmunoassay device), other electron transport inhibitors and uncouplingagents may require addition of ethanol or similar solvent to aiddissolution in the plasma fraction.

Without being bound by theory, electron transport inhibitors generallyact by binding along the electron transport chain, thus preventingelectrons from being passed from one molecule in the sequence to thenext. The mechanism of inhibition can be along the NADH pathway, thesuccinate pathway, or the common pathway. For example, cyanide is aneffective reversible inhibitor of cytochrome oxidase which is theterminal enzyme in the common pathway. Specifically, a concentration ofabout 1 mM KCN (optionally from 0.5 to 50 mM) should be sufficient tosubstantially inhibit oxygen consumption in mitochondria present in ahuman blood sample.

Uncoupling agents act by interfering with the chemiosmotic gradient. Forexample, 2,4-dinitrophenol works as a proton ionophore, binding toprotons on one side of the membrane, and being lipophilic, transportsthe protons across the membrane. Thus, the respiratory chain isprevented from maintaining a proton gradient across the membrane.Carbonyl cyanide p-[trifluoromethoxyl]-phenyl-hydrozone (FCCP) alsoworks in this manner. Oligomycin has a related effect and acts bybinding ATP synthase in such a way as to block the proton channel, thusinhibiting oxidative phosphorylation. The use of these agents is alsobeneficial for ameliorating interference based on any anaerobicleukocyte activity. Specifically, even when the cells are deprived ofoxygen, conversion of glucose to lactate still can yield ATP, unless theuncoupling agents are used.

In some embodiments of the invention, the reagent is glucose oxidase andan electron acceptor. In still other embodiments, the reagent compriseshexokinase and a source of adenosine-5′-triphosphate (ATP). In otherembodiments, the reagent comprises hexokinase, creatine kinase,adenosine diphosphate (ADP), and creatine phosphate. In someembodiments, the reagent comprises glucose dehydrogenase and in yetothers, the reagent comprises glucose dehydrogenase, NAD, NADH oxidase,and an electron acceptor. In other embodiments of the invention, thereagent is an oxygen depleting reagent. In others, the reagent is anoxygen depleting reagent selected from the group consisting ofdithionite, glucose oxidase, and ascorbate oxidase. In additionalembodiments, the reagent is a mitochondrial electron transport inhibitorand in others, the reagent is a mitochondrial membrane uncoupling agent.In other embodiments, the reagent is selected from the group consistingof cyanide, antimycin, rotenone, malonate, carbonyl cyanidep-[trifluoromethoxyl]-phenyl-hydrozone, 2,4-dinitrophenol, andoligomycin. In others, the reagent is an amphipathic reagent.

A preferred approach for reducing or eliminating phagocytic activity ofleukocytes towards an immunosensor is based on the addition of saponin,e.g., from Quillaja bark, which is an amphipathic reagent. The saponinacts as a surfactant and hemolytic agent that enhances transport ofproteins and other macromolecules through the cell membrane.

In an alternative embodiment of the invention, a leukocidin may be usedas the leukocidal reagent. Leukocidin is a type of cytotoxin frombacteria, e.g., Panton-Valentine leukocidin. Leukocidin is a poreforming toxin capable of killing leukocytes. See, for example, Hongo etal., Journal of Infectious Diseases 200, 715-723, (2009).

In another embodiment, a mucopolysaccharide, e.g., heparin, may be usedto induce leukocyte lysis. See, for example, Adachi et al, JPharmacobio-Dyn 9, 207-210 (1986).

In certain embodiments, the leukocidal reagent is an AHN-1 antibody. Inother embodiments of the invention, the leukocidal reagent islipocortin-1 or an equivalent thereof.

In still other embodiments, the leukocidal reagent as described hereincan be used in conjunction with beads opsonized for leukocytes. In onepreferred embodiment, the leukocidal reagent comprises glucose oxidaseand glucose, and this leukocidal reagent is used in conjunction withbeads opsonized for leukocytes.

In a preferred embodiment, the one or more leukocidal reagents areincorporated into a dry reagent coating, while in other embodiments, theleukocidal reagents are provided in a separate reagent fluid, e.g., in afluid pouch for mixing with the sample. If provided as a dry reagent,the one or more leukocidal reagents may be provided in the same dryreagent coating that contains the signal-generating reagent (e.g.,signal antibody for non-competitive assays or labeled analyte forcompetitive assays). Thus, in one embodiment of the invention, theanalysis device includes a dry reagent coating that comprises either orboth: (a) one or more leukocidal reagents, and/or (b) asignal-generating reagent such as a signal antibody or a labeledanalyte. The dry reagent coating may be formed from a reagent cocktail,which also preferably comprises either or both: (a) one or moreleukocidal reagents, and/or (b) a signal-generating reagent such as asignal antibody or a labeled analyte. In some embodiments, the reagentcoating and/or cocktail further comprises one or more of: (a) IgM orfragments thereof for ameliorating interference caused by heterophileantibodies, as disclosed in co-pending U.S. application Ser. No.12/411,325, referenced above, and/or (b) sacrificial beads opsonized forleukocytes (discussed below), as described in previously discussed U.S.patent applicaiton Ser. Nos. 12/620,179 and 12/620,230. The surface onwhich the reagent cocktail is to be deposited preferably is first Coronatreated to provide charged surface groups that will promote spreading ofthe printed cocktail.

In some embodiments, the reagent cocktail used to form the dry reagentcoating may further comprise one or more of a water-soluble protein, anamino acid, a polyether, a polymer containing hydroxyl groups, a sugaror carbohydrate, a salt and optionally a dye molecule. In oneembodiment, the cocktail contains bovine serum albumin (BSA), glycine,salt, methoxypolyethylene glycol, sucrose and optionally a visualizationagent such as for example bromophenol blue to provide color that aidsvisualizing the printing process. In another embodiment, from 1 to 20 μLof cocktail is printed onto the desired surface, e.g., within theholding chamber or other conduit, of the analysis device and allowed toair dry (with or without heating) before being assembled with its cover.In a preferred embodiment, the reagent cocktail and the dry reagentcoating formed therefrom comprise lactitol, DEAE-dextran, salts such asmagnesium and sodium chloride, IgG/IgM, heparin, surfactant(s) andrhodamine.

In other embodiments of the invention, the cartridge may comprise aplurality of dry reagent coatings (in which case the coatings may berespectively referred to as a first reagent coating, a second reagentcoating, etc., in order to distinguish them). For example, theleukocidal reagent may be included in a first reagent coating, which,for example, may be adjacent to a second reagent coating that containsthe signal generating element, e.g., signal antibody for non-competitiveassays or labeled analyte for competitive assays. In this aspect, thesecond reagent coating may be located upstream or downstream of thefirst reagent coating, although it is preferable for the reagent coatingthat contains the signal generating element to be located downstream ofthe reagent coating that contains the sacrificial beads. In a preferredembodiment, the holding chamber is coated with a first reagent coatingthat comprises the leukocidal reagent and optionally other reagents,e.g., sacrificial beads, that ameliorate various forms of interference.In this aspect, a second reagent coating comprising the signalgenerating element preferably is located downstream of the holdingchamber, e.g., immediately upstream of the immunosensor.

In some embodiments, a reagent cocktail preferably is formulated as aprintable aqueous solution containing the leukocidal reagent andoptionally other interference-reducing reagents, e.g., sacrificialbeads. Upon introduction of a biological sample, e.g., blood, the samplepreferably mixes with the reagent in a first step of the assay. In someembodiments, the reagent may also include inorganic salts andsurfactants to optimize assay performance with respect to chemical andfluidic attributes. In further embodiments, the reagent may includeoptional additives such as an anticoagulant or anticoagulation agent(e.g., heparin) to ensure adequate anticoagulation and/or avisualization agent (e.g., a dye or colorant) for visualization of thelocation of the reagent after printing. Also optionally present arestabilizers such as sodium azide for inhibition of microbial growth anda mixture of lactitol and diethylaminoethyl-dextran (Applied EnzymeTechnologies Ltd., Monmouth House, Mamhilad Park, Pontypool, NP4 0HZ UK)for stabilization of proteins. Once deposited in the device, thedeposited reagent may, for example, be dried for 30 to 60 minutes in astream of warm air. In one embodiment, the reagent is printed in thesample inlet of the device using an automated printing instrument anddried to form a leukocidal reagent-containing coating layer.

IV. Sacrificial Beads

As indicated above, in addition to the use of one or more leukocidalreagents, the sample, e.g., blood sample, may be further amended withsacrificial or decoy beads that are homogeneously mixed with the sample,and where the beads are specifically opsonized with respect toleukocytes.

In one embodiment of the invention, the sacrificial beads are coatedwith a non-human IgG (IgG class immunoglobulins) or fragments thereofisolated from animal species. As used herein, the term “fragment” refersto any epitope-bearing fragment derived from the specified molecule.Thus, an IgG fragment may comprise, for example, epitope bearing F(ab′)2or Fab fragments or an Fc fragment. In addition, the phrase “IgG orfragments thereof” is meant to include IgG alone, IgG fragments alone(i.e., one or more of F(ab′)2 fragments, Fab fragments and/or Fcfragments of IgG), or a combination of IgG and IgG fragments. Thedesired effect may be achieved with a variety of surface coatings, solong as the surface coating is opsonized or opsonizable upon exposure toa sample to be assayed.

As indicated above, in a preferred embodiment, the sacrificial beads areincorporated into a dry reagent coating, which in some embodiments maybe the same dry reagent coating that contains the signal-generatingreagent (e.g., signal antibody for non-competitive assays or labeledanalyte for competitive assays) or the leukocidal reagent. Thus, in oneembodiment of the invention, the analysis device includes a dry reagentcoating that comprises either or both: (a) a leukocidal reagent; (b) acomponent suitable for ameliorating the effect of leukocytes, e.g.,beads coated with IgG or fragments thereof, and/or (c) asignal-generating reagent such as a signal antibody or a labeledanalyte.

V. Additives

In some embodiments of the invention, additives may be included in thecartridge or used in conjunction with the assay. In certain embodiments,an anticoagulant can be added. For example, in some embodiments, heparinis added to improve performance in cases where the sample was notcollected in a heparinized tube or was not properly mixed in aheparinized tube. A sufficient amount of heparin is added so that freshunheparinized blood will remain uncoagulated during the assay cycle ofthe cartridge, typically in the range of 2 to 20 minutes. In otherembodiments, goat and mouse IgG can be added to combat heterophileantibody problems known in the immunoassay art. In still otherembodiments, one or more of proclin, DEAE-dextran, tris buffer, andlactitol can be added as reagent stabilizers. In other embodiments, asurfactant such as for example polysorbate 20 (Tween-20) can be added toreduce binding of proteins to the plastic, which is the preferredmaterial for the cartridge. The addition of a surfactant alsofacilitates the even coating of reagents on the plastic surface and actsas an impurity that minimizes the crystallization of sugars, such aslactitol. In other embodiments of the invention, a antibacterial agentor biocide (e.g., sodium azide) may be added to inhibit bacterialgrowth.

VI. Preparation of Print Cocktail and Printing

In a preferred embodiment, the base print cocktail is prepared asfollows for a 1 liter (L) batch: protein stabilization solution (PSS,AET Ltd., 50% solids, 100.0 g) is added to 200-250 mL of an aqueoussolution of sodium chloride (8.00 g) and sodium azide (0.500 g) and theresulting solution is transferred to a 1 L volumetric flask. A solutionof murine IgG is prepared by adding murine IgG (0.9 g) to 75 mL ofdeionized water and stirred for 15-60 minutes until dissolution iscomplete. An equally concentrated solution of caprine IgG is prepared inan identical manner and both solutions are filtered through a 1.2 μMfilter. Murine IgM is acquired as a liquid from a supplier (for example,Sigma-Aldrich). The protein concentrations of each of the threeimmunoglobulin (Ig) stock solutions are measured spectrophotometricallyat 280 nm. The masses of these Ig solutions required to provide murineIgG (0.75 g), caprine IgG (0.75 g), and murine IgM (25 mg) arecalculated and these amounts are added to the printing solution. Asolution of diethylaminoethyl-dextran (DEAE-dextran) is prepared byadding DEAE-dextran (2.5 g) to 50-100 mL of deionized water and stirringfor 30 minutes. The DEAE-dextran solution is added to the printingsolution. To this is added sodium heparin (10,000 IU/mL, 3.00 mL),Tween-20 (3.00 g) and a 5% (w/v) aqueous solution of rhodamine (200 μL).The resulting solution is diluted to 1.000 L with deionized water andstored in a freezer or refrigerator until use. When included, IgG-coatedmicroparticles for leukocyte interference mitigation can be added beforethis final dilution. Likewise leukocidal reagents of the type describedabove may be added before the final dilution or just prior to depositioninto a test device, e.g., a cartridge of the general type shown in FIGS.1-6.

In various embodiments of the invention, printing of print cocktails andsimilar fluids to form a dry reagent coating on the cartridge componentis preferably automated and based on a microdispensing system, includinga camera and computer system to align components, as disclosed in U.S.Pat. No. 5,554,339 to Cozzette et al. (the “'339 Patent”), which isincorporated herein by reference in its entirety. In the '339 Patent,the wafer chuck is replaced by a track for feeding the plastic cartridgebases to the dispensing head. The track presents the bases to the headin a predetermined orientation to ensure consistent positionaldispensing.

VII. Collection and Analysis

According to various embodiments of the invention, the leukocidalreagent, and (if employed) sacrificial beads may be incorporated in asample collection device, e.g., capillary, Vacutainer™ or syringe. Insome embodiments, the leukocidal reagent coating is formed on aninterior wall of the collection device. As such, in one embodiment, theinvention is to a kit for performing an immunoassay that comprises theleukocidal reagent, which is first used to amend the blood sample in afirst container or location, and then the sample is passed to a secondcontainer or location which has the capture and signal antibodies.

In another embodiment, the leukocidal reagent may be contained insolution and mixed with the biological sample, e.g., blood, and theresulting amended sample is introduced into the analysis device, e.g.,cartridge. In one embodiment, for example, a blood sample may be mixedwith the leukocidal reagent (and optionally sacrificial beads) to forman amended sample, which is then introduced into the analysis device,e.g., cartridge. In another aspect, the device includes a pouch thereinthat contains a liquid comprising the leukocidal reagent and optionallysacrificial beads, which may be mixed with a blood sample in the deviceand then processed substantially as described herein to form an assay,e.g., sandwich assay, for analyte detection.

In still another embodiment, electrowetting is employed to mix a firstliquid comprising the leukocidal reagent with a liquid biological samplesuch as for example blood. In this embodiment, an apparatus may beprovided for manipulating droplets. The apparatus, for example, may havea single-sided electrode design in which all conductive elements arecontained on one surface on which droplets are manipulated. In otherembodiments, an additional surface can be provided parallel with thefirst surface for the purpose of containing the droplets to bemanipulated. The droplets are manipulated by performingelectrowetting-based techniques in which electrodes contained on orembedded in the first surface are sequentially energized andde-energized in a controlled manner. The apparatus may allow for anumber of droplet manipulation processes, including merging and mixingtwo droplets together, splitting a droplet into two or more droplets,sampling a continuous liquid flow by forming from the flow individuallycontrollable droplets, and iterative binary or digital mixing ofdroplets to obtain a desired mixing ratio. In this manner, droplets ofthe first liquid comprising the leukocidal reagent and other reagents(e.g., sacrificial beads) may be carefully and controllably merged andmixed with the liquid biological sample, e.g., blood. (See, e.g., U.S.Pat. No. 6,911,132 to Pamula et al., the entirety of which isincorporated herein by reference).

VIII. Immunosensor Embodiments and Methods Related Thereto

While the present invention is broadly applicable to immunoassaysystems, it is best understood in the context of the i-STAT® immunoassaysystem (Abbott Point of Care Inc., Princeton, N.J., USA), as describedin the jointly-owned pending and issued patents cited above. See alsoU.S. Patent Application No. 61/288,189, entitled “Foldable CartridgeHousings for Sample Analysis,” filed Dec. 18, 2009, the entirety ofwhich is incorporated herein by reference. In various embodiments, thesystem employs an immuno-reference sensor (See, e.g., U.S. Pat. Appl.Pub. 2006/0160164 to Miller et al., referenced above and incorporatedherein by reference in its entirety) for purposes of assessing thedegree of non-specific binding (NSB) occurring during an assay. NSB mayarise due to inadequate washing or due to the presence of interferences.The net signal from the assay is comprised of the specific signalarising from the analyte immunosensor corrected by subtracting thenon-specific signal arising from the immuno-reference sensor. The amountof signal at the immuno-reference sensor is subject to limits defined bya quality control algorithm.

Embodiments of the present invention improve the resistance of thei-STAT® immunoassay format to interference caused by leukocytes;however, such embodiments are equally applicable to the standard ELISAformat, where cells are present in the analysis medium. In someembodiments, the sample is amended with sacrificial beads preferably incombination with a leukocidal reagent of the present invention toprovide reduced or eliminated leukocyte interference.

It should be noted that while traditional sandwich assays may yielderroneous results for biological samples having a high leukocyte level,prior i-STAT® system assays did not previously report inaccurate resultsfor these samples, as the system includes an algorithm that detectsspurious signals, alerts the user with an error code, and suppresses theresult from being displayed. This is one embodiment of a quality systemfor reliable point-of-care testing. In this way the quality andintegrity of the analytical system is maintained.

Surprisingly and unexpectedly, when modified cartridges containingleukocidal reagents were tested and the results compared withconventional cartridges lacking these materials, the resultsdemonstrated that blood samples exhibiting leukocyte interference in theconventional cartridges could be analyzed accurately when the leukocidalreagents were employed.

With regard to the subject matter of the present invention, it wasdiscovered that certain immunoassay test cartridges, notably BNPcartridges, exhibited unexpected positively and negatively slopingwaveforms arising from a previously unknown interference mechanism. See,for example, FIGS. 11A and B, respectively. This led to a hypothesisconcerning the mechanism of the interference based on experiments on theeffect of pulsing the electrode to positive potentials during the washstep. As shown in FIGS. 11C and D, the normal immunosensor response,expected on a theoretical basis, has a near-zero slope at low analyteconcentrations, and negative slopes are expected only at high analyteconcentrations where the measurement can become substrate-limited ratherthan enzyme limited.

There are several potential mechanisms causing dynamic (non-steadystate) amperometric signals. Dynamic electrode activity (changingeffective electrode area) is unlikely since the electrode is sequesteredfrom formed elements owing to the presence of immobilized assay beads(microparticles) on a polyvinyl alcohol (PVA) layer, neither of whichexhibit this effect in plasma samples. Dynamic layer thickness, e.g.,swelling or shrinking of the components above, depending on fluidcontact, is unlikely as there is no driving force for such a phenomenonthat would elicit both positive and negative slopes. Dynamic coverage ofthe enzyme (e.g., ALP, changing surface concentration of enzyme) isruled out because in the thin-layer format, diffusion of enzyme (e.g.,ALP) from the sensor over the time-scale of the measurement is notpossible. Dynamic transport of the enzyme substrate, e.g.,para-aminophenol phosphate (p-APP), to the electrode surface is unlikelygiven the size of the molecule, which is relatively small and has arelatively facile diffusion (D≈5×10⁻⁶ cm²/s). Thus, by the process ofelimination, dynamic ALP activity was considered the most likely causeof dynamic sensor signals and further investigation was made to assessthe mechanism. It is noted that the hydrolysis of p-aminophenylphosphate by ALP is pH-dependent with an optimum near pH 10. In someembodiments, in the cartridge, a working pH of 9.2 is used as thisslightly lower pH value ensures stability of immunocomplexes. Inaddition, the oxidation of para-aminophenol (p-AP) at the electrode ispH dependent and is expected to shift by about +59 mV per decadedecrease in pH, i.e., at lower pH the reaction must be driven harder,i.e., an increased applied potential is required.

In embodiments where the microparticle print layer was to become lesspermeable due to interaction with the interfering components to theextent that sample fluid (pH 7.4) within the sensor structure could notbe fully replaced with analysis fluid (pH 9.2) during the wash step,this results in suboptimal detection step pH, particularly for theelectrode reaction which occurs at a region farthest from the point ofentry of the analysis fluid. This impaired fluid replacement creates apH gradient that undergoes relaxation with time. As the relative ratesof the enzyme and electrode reactions change, so will the observedsignal. This technical evaluation has the considerable advantage that itoffers an explanation for both positive and negatively sloping signals.See, e.g., FIGS. 11A and B.

The conception that the observed interference is associated with anincomplete wash step due to “plugging” or “fouling” of the microparticlelayer was assessed further by applying a pulse to extreme potentials,e.g., applying a pulse to an oxidizing potential, during the wash step.It was anticipated that positively sloping waveforms might be caused byan inactive blocked sensor and that pulsing might be employed to cleanthe electrode prior to the analysis step. It was observed that applyingan oxidizing pulse during the wash step in a normal sample had no effecton observed signal and in the subsequent analysis. See FIG. 12A.However, in the case of aberrant high buffy samples the effect ofpulsing was large and dynamic signals and further, these signals weregreater than expected given the concentration of analyte. See FIG. 12B.

The difference in the response to an oxidizing potential pulse in thesetwo cases demonstrated that the fluids within the sensor structure wereindeed different. As anticipated, the correct response resulted from thepresence of desired analysis fluid over the sensor, whereas the abnormalresponse arose because the fluid over the sensor structure was plasma,or a combination of plasma and analysis fluid. The difference inresponse can be understood as follows: the oxidizing pulse results inthe evolution of oxygen and a decrease in pH according to: H₂O→ 1/2O₂+2H⁺+2e ⁻.

In the case of the sensor structure containing analysis fluid bufferedto pH 9.2, protons evolved at the electrode were rapidly consumed in thepresence of the buffer (100 mM carbonate in analysis fluid). However, inthe absence of the buffering afforded by the analysis fluid, protonspersisted in the region of the electrode resulting in a plume of acidicfluid. This acidic plume resulted in the acid-catalyzed hydrolysis ofpAPP to pAP generating a larger signal than anticipated. Specifically,the aberrant waveform arose from pAP generated by the enzymatic actionof the enzyme, e.g., ALP, on pAPP and also non-enzymatic acid-catalyzedhydrolysis.

It is noted that the acid-catalyzed hydrolysis reaction does not occursignificantly for p-aminophenyl phosphate unless the amino group isprotonated as it is at low pH. This is because the reaction requires astrongly electron-withdrawing substituent in the para position (Barnardet al., J. Chem. Soc. (1966), 227-235). As is known in the art, an —NH₂substituent is significantly electron-donating, whereas upon protonationthe —NH₃ ⁺ substituent becomes highly electron-withdrawing (more so eventhan —NO₂; for example the Hammet para-rho values are −0.66 for —NH₂ and1.70 for —NH₃ ⁻).

These experiments associated the observed interference with “high buffy”samples and could intentionally be elicited by running whole bloodsamples with an enriched buffy coat. This term is given to the layer ofwhite blood cells and platelets that form at the plasma-red cellboundary when a blood sample is centrifuged. Further evidenceimplicating leukocytes and potentially platelets as fouling agents isshown in the micrographs FIGS. 17A (assay without use of sacrificialbeads or leukocidal reagent) and 17B (assay after using sacrificialbeads). For comparison, a pristine immunosensor prior to contact with ablood sample is illustrated in FIG. 20. Similar results were obtainedproviding visual confirmation for the use of leukocidal reagents inreducing leukocyte adhesion to sensors.

FIG. 17A shows a sensor that was exposed to a high buffy sample (˜10⁵leukocytes per μL) using the standard measurement cycle with a sensorincubation time of 10 minutes. The sample was not exposed to opsonizedsacrificial beads or any leukocidal reagents. It is clear that a portionof the assay bead-coated sensor surface is covered with an adhered layerof cells that were not easily removed by the wash step.

By contrast, FIG. 17B shows a sensor that was exposed to a high buffysample (˜10⁵ leukocytes per μL) using the standard measurement cyclewith a sensor incubation time of 10 minutes. However, in contrast to theassay shown in FIG. 17A, the sample shown in FIG. 17B was exposed toopsonized (IgG coated) sacrificial beads. It is clear that the portionof the assay bead-coated sensor surface had significantly less adheredcells when compared to FIG. 17A after the wash step.

IgG acts as an opsonin, which is a substance capable of marking apathogen for phagocytosis, for example, by leukocytes. IgGs aregenerally added to immunoassays to manage heterophile antibodyinterference as described in jointly-owned pending U.S. application Ser.No. 12/411,325, and are present on assay beads in the BNP cartridgedescribed herein. Consequently, it is likely that either this source ofIgG (when present in an immunoassay device) or IgG naturally present inthe blood sample may act to undesirably opsonize the sensor surface toleukocytes. In addition, as the assay beads are similar in size tobiological cells (bacteria), which are the natural target ofphagocytosis, it is probable that IgG accumulation on the assay beads isundesirably promoting accumulation of leukocytes on these beads. This isconsistent with the observed interference in samples with high whitecell counts, and possibly those with an activated immune status. Thepresent invention provides a solution to this leukocyte interferencewhereby the amendment of a sample with a leukocidal reagent, optionallywith sacrificial IgG-coated microparticles, affords a means fordecreasing leukocyte activity so as to divert them from the primaryimmune reagents on the sensor.

As indicated above, in some embodiments, the sample is amended withsacrificial beads in addition to the leukocidal reagent. Preparation ofIgG-coated microparticles may be effected using methods analogous tothose employed for preparation of the assay beads. This method involvesadsorption of IgG onto carboxylated polystyrene microparticles in MESbuffer (2-(N-morpholino)ethanesulfonic acid) followed by cross-linkingin the presence of EDAC (1-ethyl-3-(3-dimethylaminopropyl)carbodiimide).

One non-limiting method for forming the sacrificial beads is nowdescribed. In a preferred procedure, raw microparticles are pelleted bycentrifugation at 18,000 RCF (relative centrifugal force) for 20 minutesfrom their matrix (10% microparticles in water, Seradyn) andre-suspended in 25 mM MES buffer to a concentration of 100-200 mg/mLmicroparticles. IgG dissolved in 25 mM MES buffer is then added in aquantity equal to about 1 to 5% of the weight of the microparticles.After a 15-20 minutes nutation in a refrigerator, the microparticles arepelleted by centrifugation for 20 minutes at 1300 RCF is re-suspended infresh MES buffer to a concentration of 75 mg/mL. The supernatant fromcentrifugation is assayed for protein content by measuring absorbance at280 nm (effective extinction coefficient of 1.4 AU/mg/mL protein) toconfirm the IgG has absorbed onto the microparticles. Freshly preparedEDAC cross-linking agent (10 mM in MES buffer) is added to there-suspended microparticles to a final concentration of about 2-4 mM.The mixture is then stirred by nutation in a refrigerator for 120±15minutes. The microparticles may then again be pelletized bycentrifugation at 1300 RCF for 20 minutes and re-suspended in 1/5 PPS(phosphate buffered saline) and nutated in the refrigerator for 15-30minutes. Upon final centrifugation, the microparticles can bere-suspended in PBS+0.05% Sodium Azide or in 1:1 1/5 PPB:PSS (1/5 PPB isPBS diluted with 4 parts water, PSS=protein stabilization solution,Applied Enzyme Technologies, Ponypool, UK) to a concentration of 10%solids. The resulting preparation may be aliquoted and stored frozen,preferably at −60° C. Microparticles suspended in PBS formed by thisprocess were employed in the experiments described herein and were doseddirectly into blood samples.

Preparation of an enriched or high buffy whole blood sample, asdescribed for use in the experiments described herein, was as follows.Fresh whole blood was drawn from a donor into two 6 mLEDTA-anticoagulated Vacutainers™ and was centrifuged for 10 minutes at2000 RCF (standard rotor). Where BNP “positive” samples were desired,the tubes were spiked with the BNP antigen prior to centrifugation. Allof the plasma except the last millimeter above the plasma/buffy coat/redblood cell interface was withdrawn and set aside. The interfacial regionwas then withdrawn using a pipettor (with the intention of removing asmuch of the buffy coat layer as possible) and was set aside. The redblood cells were then removed and set aside. By recombining the buffycoat materials with lesser portions of the plasma and red cellfractions, it was possible to create high buffy blood samples whileretaining a normal sample hematocrit, e.g., 35-55 wt. %. Similarly,samples with a low or essentially no buffy material (leukocytes andplatelets) present can be created.

In experiments, BNP test cartridges were tested with: (i) high buffysamples, (ii) high buffy samples treated with a 10 percent volume asuspension of 10 weight percent microparticles coated with IgG (μP-IgG)in PBS immediately before running, (iii) high hematocrit leukocyte-freesamples, and (iv) low hematocrit leukocyte-free samples.

FIG. 21 contains graphical data illustrating the effect of leukocyteinterference on electrochemical immunosensor signal slopes and theeffect of sacrificial microparticle treatment on the slopes. Illustratedin the right and left panels are Sensor Slopes (y-axis) plotted as afunction of Net Signal (x-axis) for normal whole blood sample (rightpanel) and high buffy blood sample (enriched leukocyte, high buffy, leftpanel). The left panel illustrates the considerable variability ofsignal slopes observed in high buffy samples in the absence ofsacrificial microparticles (Sublot 1). This variability wassubstantially ameliorated in the presence of sacrificial microparticles(Sublots 2 and 3). Minimal signal slope variability was observed in thenormal whole blood samples (right panel) both with and withoutsacrificial microparticles.

Microscopic inspection of sensors following the assay revealed thepresence of a thick deposit on chips run in high buffy (HB) and theabsence of a deposit on samples run in HB/μP-IgG. Micrographs of theimmunosensors are shown in FIGS. 17A and 17B and their associatedanalyzer waveforms are illustrated in FIGS. 22A and B, respectively. Itis clear from a comparison of a pristine immunosensor prior to contactwith a blood sample as illustrated in FIG. 20 with the immunosensorafter contact with blood treated with the sacrificial beads (FIG. 17B)that visually there is a significant improvement, i.e., a reduction inadhered leukocytes compared to FIG. 17A. Based on many observations,this visual improvement correlates directly with an actual improvementin immunosensor performance.

Additional experiments showed that the interference phenomenon does notoccur in plasma alone or in samples containing platelets but notleukocytes, but only in samples containing a high buffy level. Inaddition, smaller microparticles, e.g., those having an average particlesize less than 0.2 μm, coated with IgG do not have the same interferencemitigating effect on sensor slopes as do larger particles. Preferably,the average particle size of the IgG-coated microparticles (sacrificialbeads), if included with the leukocidal reagent(s), is from 0.01 to 20μm, e.g., from 0.1 to 20 μm, from 1 to 10 μm, from 0.1 to 5 μm, or from2 to 5 μm. The particle size distribution of the sacrificialmicroparticles preferably is unimodal, although polymodal distributionsare also possible. In principal, any particles of the correct size andcapable of being opsonized may be used; however, polystyrene beads arepreferred. In preferred embodiments of the invention, goat or sheep IgGcoated particles are employed, although other IgG sources may beemployed such as, for example, mouse or rabbit IgG. In general, it wasfound that the size of the microparticles is important, but not itscomposition or the source of the IgG. With regard to the sacrificialbeads, the beads, if employed, preferably comprise substrate beadsformed of a material selected from the group consisting of polystyrene,polyacrylic acid and dextran, and can have an average particle size inthe range of about 0.01 μm to about 20 μm, more preferably an averageparticle size in the range of from 0.1 μm to 5 μm or from about 2 μm toabout 5 μm. While use of a spherical bead is preferred, in otherembodiments, other bead shapes and structures, e.g., ovals,sub-spherical, cylindrical and other irregular shaped particles, arewithin the meaning of the terms “beads” and “microparticles” as used anddefined herein. As used herein, the term “average particle size” refersto the average longest dimension of the particles, for example thediameter for spherical particles, as determined by methods well-known inthe art.

Collectively, the experimental data support the conclusion thatleukocytes are primarily responsible for a phenomenon in which thesensor becomes less permeable in the wash cycle and that thisimmunoassay interference can be ameliorated by addition of sacrificialIgG coated particles to the assay medium.

Wafer-level microfabrication of a preferred embodiment of theimmunosensor of the present invention is as follows. As shown in FIG. 9,the base electrode 94 comprises a square array of 7 μm gold disks on 15μm centers. The array covers a circular region approximately 600 μm indiameter, and is achieved by photo-patterning a thin layer of polyimideof thickness 0.35 μm over a substrate made from a series of layerscomprising Si/SiO₂/TiW/Au. The array of 7 μm microelectrodes affordshigh collection efficiency of electroactive species with a reducedcontribution from any electrochemical background current associated withthe capacitance of the exposed metal. The inclusion of a poly(vinylalcohol) (PVA) layer over the metal significantly enhances the reductionof background currents.

In some embodiments, the porous PVA layer is prepared by spin-coating anaqueous mixture of PVA plus a stilbizonium photoactive, cross-linkingagent over the microelectrodes on the wafer. The spin-coating mixtureoptionally includes bovine serum albumin (BSA). The wafer is thenphoto-patterned to cover only the region above and around the arrays andpreferably has a thickness of about 0.6 μm.

The general concept of differential measurement is known in theelectrochemical and sensing arts. A novel means for reducing interferingsignals in an electrochemical immunosensing systems is now described.However, while it is described for pairs of amperometric electrochemicalsensors it is of equal utility in other electrochemical sensing systemsincluding, but not limited to potentiometric sensors, field effecttransistor sensors and conductimetric sensors. Embodiments of thepresent invention are also applicable to optical sensors, e.g.,evanescent wave sensors and optical wave guides, and also other types ofsensing including acoustic wave and thermometric sensing and the like.Thus, according to various embodiments of the invention, the immobilizedantibody may be attached to a sensor selected from the group consistingof an amperometric electrode, a potentiometric electrode, aconductimetric electrode, an optical wave guide, a surface plasmonresonance sensor, an acoustic wave sensor and a piezoelectric sensor.Ideally, in the non-competitive assay embodiments, the signal from animmunosensor (IS) is derived solely from the formation of a sandwichcomprising an immobilized antibody (Ab1), the analyte, and a signalantibody (Ab2) that is labeled, wherein the label (e.g., an enzyme)reacts with a substrate (S) to form a detectable product (P) as shownbelow in scheme (1).Surface-Ab1-analyte-Ab2-enzyme; enzyme+S→P   (1)

It is known that some of the signal antibody (Ab2) may bindnon-specifically to the surface, as shown below in schemes (2) and (3),and might not be washed away completely from the region of theimmunosensor (up to approx. 100 microns away) during the washing stepthereby giving rise to a portion of the total detected product that isnot a function of the surface-Ab1-analyte-Ab2-enzyme immunoassaysandwich structure, thereby creating an interfering signal.Surface-Ab2-enzyme; enzyme+S→P   (2)Surface-analyte-Ab2-enzyme; enzyme+S→P   (3)

As indicated above, a second immunosensor optionally may be placed inthe cartridge that acts as an immuno-reference sensor (IRS) and givesthe same (or a predictably related) degree of NSB as occurs on theprimary immunosensor. In one embodiment, interference can be reduced bysubtracting the signal of this immuno-reference sensor from that of theprimary immunosensor, i.e., the NSB component of the signal is removed,improving the performance of the assay, as shown in scheme (4) below. Inanother embodiment, the correction may optionally include thesubtraction or addition of an additional offset value.Corrected signal=IS−IRS   (4)

In preferred embodiments of the invention, the reference immunosensor,also referred to as immuno-reference sensor, is the same in allsignificant respects (e.g., dimensions, porous screening layer, latexparticle coating, and metal electrode composition) as the primaryimmunosensor except that the capture antibody for the analyte (e.g.,cTnI) is replaced by an antibody to a plasma protein that naturallyoccurs in samples (both normal and pathological) at a highconcentration. The immunosensor and reference immunosensor may befabricated as adjacent structures 94 and 96, respectively, on a siliconchip as shown in FIG. 9. While the preferred embodiment is described fortroponin I and BNP assays, this structure is also useful for othercardiac marker assays including, for example, troponin T, creatinekinase MB, procalcitonin, proBNP, NTproBNP, myoglobin and the like, plusother sandwich assays used in clinical diagnostics, e.g., PSA, D-dimer,CRP, HCG, NGAL, myeloperoxidase and TSH.

Examples of suitable antibodies that bind to plasma proteins includeantibodies to human serum albumin, fibrinogen and IgG fc region, withalbumin being preferred. However, any native protein or blood componentthat occurs at a concentration of greater than about 100 ng/mL can beused if an appropriate antibody is available. The protein should,however, be present in sufficient amounts to coat the sensor quicklycompared to the time needed to perform the analyte assay. In a preferredembodiment, the protein is present in a blood sample at a concentrationsufficient to bind more than 50% of the available antibody on thereference immunosensor within about 100 seconds of contacting a bloodsample. In general the second immobilized antibody has an affinityconstant of about 1×10⁻⁷ to about 1×10⁻¹⁵ M. For example, an antibody toalbumin having an affinity constant of about 1×10⁻¹⁰ M is preferred, dueto the high molar concentration of albumin in blood samples, which isabout 1×10⁻⁴ M.

According to various embodiments of the present invention, providing asurface that is covered by native albumin derived from the samplesignificantly reduces the binding of other proteins and cellularmaterials that may be present. This method is generally superior toconventional immunoassays that use conventional blocking agents tominimize NSB because these agents must typically be dried down andremain stable for months or years before use, during which time they maydegrade, creating a stickier surface than desired and resulting in NSBthat rises with age. In contrast, the embodiments of the presentinvention provide for a fresh surface at the time of use.

An immunosensor for cardiac brain natriuretic peptide (BNP) with areference immunosensor for performing differential measurement to reducethe effect of NSB is described as follows. In one embodiment,carboxylate-modified latex microparticles (supplied by BangsLaboratories Inc., Fishers, Ind., USA or Seradyn Inc., Indianapolis,Ind., USA) coated with anti-BNP and anti-HSA are both prepared by thesame method. The particles are first buffer exchanged by centrifugation,followed by addition of the antibody, which is allowed to passivelyadsorb onto the particles. The carboxyl groups on the particles are thenactivated with EDAC in MES buffer at pH 6.2, to form amide bonds to theantibodies. Any bead aggregates are removed by centrifugation and thefinished beads are stored frozen.

In another embodiment, the anti-human serum albumin (HSA) antibody,saturation coverage of the latex beads results in about a 7% increase inbead mass. In yet another embodiment, coated beads are prepared usingcovalent attachment from a mixture comprising 7 mg of anti-HSA and 100mg of beads. Using this preparation, a droplet of about 0.4 nL,comprising about 1% solids in deionized water, is microdispensed (usingthe method and apparatus of U.S. Pat. No. 5,554,339, referenced aboveand incorporated herein by reference in its entirety) onto aphoto-patterned porous PVA permselective layer covering sensor 96, andis allowed to dry. The dried particles adhere to the porous layer andsubstantially prevent their dissolution in the blood sample or thewashing fluid.

In one embodiment of the invention, for the BNP antibody, saturationcoverage of the latex bead surface results in a mass increase in thebeads of about 10%. Thus by adding 10 mg of anti-BNP to 100 mg of beadsalong with the coupling reagent, saturation coverage was achieved. Thesebeads were then microdispensed onto sensor 94.

In another embodiment, immunosensor 94 is coated with beads having botha plasma protein antibody, e.g., anti-HSA, and the analyte antibody,e.g., anti-BNP. Latex beads made with the about 2 mg or less of anti-HSAper 100 mg of beads saturation-coated with anti-BNP provide superior NSBproperties at the immunosensor. It has been found that the slope (signalversus analyte concentration) of the troponin assay is not materiallyaffected because there is sufficient anti-BNP on the bead to capture theavailable analyte (antigen). By determining the bead saturationconcentration for different antibodies, and the slope of an immunosensorhaving beads with only the antibody to the target analyte, appropriateratios of antibody combinations can be determined for beads havingantibodies to both a given analyte and a plasma protein.

An important aspect of immunosensors having a reference immunosensor isthe “humanizing” of the surface created by a layer of plasma protein,preferably the HSA/anti-HSA combination. This surface “humanizing”appears to make the beads less prone to NSB of the antibody-enzymeconjugate and also seems to reduce bead variability. Without being boundby theory, it appears that as the sensors are covered by the sample theyare rapidly coated with native albumin due to the anti-HSA surface. Thisgives superior results compared to conventional blocking materials,which are dried down in manufacturing and re-hydrated typically after along period in storage. Another advantage to “humanizing” the sensorsurface is that it provides an extra mode of resistance to humananti-mouse antibodies (HAMA) and other heterophile antibodyinterferences. The effects of HAMA on immunoassays are well known.

In another embodiment, the immuno-reference sensor is employed in thedevices and methods of the invention to monitor the wash efficiencyobtained during the analytical cycle. As stated above, one source ofbackground noise is the small amount of enzyme conjugate still insolution, or non-specifically absorbed on the sensor and not removed bythe washing step. This aspect of the invention relates to performing anefficient washing step using a small volume of washing fluid, byintroducing air segments as mentioned in Example 2.

In operation of the preferred embodiment, which is an amperometricelectrochemical system, the currents associated with oxidation ofp-aminophenol at immunosensor 94 and immuno-reference sensor 96 arisingfrom the activity of ALP, are recorded by the analyzer. The potentialsat the immunosensor and immuno-reference sensor are poised at the samevalue with respect to a silver-silver chloride reference electrode. Toremove the effect of interference, the analyzer subtracts the signal ofthe immuno-reference sensor from that of the immunosensor according toequation (4) above. Where there is a characteristic constant offsetbetween the two sensors, this value is also subtracted. It is notnecessary for the immuno-reference sensor to have all of the samenon-specific properties as the immunosensor, only that theimmuno-reference sensor be consistently proportional in both the washand NSB parts of the assay. In one embodiment, an algorithm embedded inthe analyzer can account for any other essentially constant differencebetween the two sensors.

Use of a differential combination of immunosensor and immuno-referencesensor, rather than an immunosensor alone, provides the followingimprovement to the assay. In a preferred embodiment, the cartridgedesign provides dry reagent that yields about 4-5 billion enzymeconjugate molecules dissolved into about a 10 μL blood sample. At theend of the binding and wash steps the number of enzyme molecules at thesensor is about 70,000. In experiments with the preferred embodimentthere were, on average, about 200,000 (±about 150,000) enzyme moleculeson the immunosensor and the reference immunosensor as non-specificallybound background. Using a differential measurement with theimmuno-reference sensor, about 65% of the uncertainty was removed,significantly improving the performance of the assay. While otherembodiments may have other degrees of improvement, the basis for theoverall improvement in assay performance remains.

An additional use of the optional immuno-reference sensor is to detectanomalous sample conditions, such as for example improperlyanti-coagulated samples which deposit material throughout the conduitsand cause increased currents to be measured at both the immunosensor andthe immuno-reference sensor. This effect is associated with bothnon-specifically adsorbed enzyme and enzyme remaining in the thin layerof wash fluid over the sensor during the measurement step.

Another use of the optional immuno-reference sensor is to correctsignals for washing efficiency. In certain embodiments, the level ofsignal at an immunosensor depends on the extent of washing. For example,longer washing with more fluid/air segment transitions can give a lowersignal level due to a portion of the specifically bound conjugate beingwashed away. While this may be a relatively small effect, e.g., lessthan 5%, correction can improve the overall performance of the assay.Correction may be achieved based on the relative signals at the sensors,or in conjunction with a conductivity sensor located in the conduitadjacent to the sensors, acting as a sensor for detecting and countingthe number of air segment/fluid transitions. This provides the input foran algorithmic correction means embedded in the analyzer.

In another embodiment of the reference immunosensor with an endogenousprotein, e.g., HSA, it is possible to achieve the same goal by having animmuno-reference sensor coated with antibody to an exogenous protein,e.g., bovine serum albumin (BSA). In this case the step of dissolving aportion of the BSA in the sample, provided as an additional reagent,prior to contacting the sensors is needed. This dissolution step can bedone with BSA as a dry reagent in the sample holding chamber of thecartridge, or in an external collection device, e.g., a BSA-coatedsyringe. This approach offers certain advantages, for example theprotein may be selected for surface charge, specific surface groups,degree of glycosylation and the like. These properties may notnecessarily be present in the available selection of endogenousproteins.

In addition to salts, other reagents can improve whole-blood precisionin an immunoassay. These reagents should be presented to the bloodsample in a way that promotes rapid dissolution. In some embodiments,support matrices including cellulose, polyvinyl alcohol (PVA), andgelatin (or mixtures thereof) coated onto the wall of the blood-holdingchamber (or another conduit) promote rapid dissolution, e.g., greaterthan 90% complete in less than 15 seconds.

A cartridge of the present invention has the advantage that the sampleand a second fluid can contact the sensor array at different timesduring an assay sequence. The sample and the second fluid also may beindependently amended with other reagents or compounds present initiallyas dry coatings within the respective conduits. Controlled motion of theliquids within the cartridge further permits more than one substance tobe amended into each liquid whenever the sample or fluid is moved to anew region of the conduit. In this way, provision is made for multipleamendments to each fluid, greatly extending the complexity of automatedassays that can be performed, and therefore enhancing the utility of thepresent invention.

In a disposable cartridge, the amount of liquid contained is preferablykept small to minimize cost and size. Therefore, in the presentinvention, segments within the conduits may also be used to assist incleaning and rinsing the conduits by passing the air-liquid interface ofa segment over the sensor array or other region to be rinsed at leastonce. It has been found that more efficient rinsing, using less fluid,is achieved by this method compared to continuous rinsing by a largervolume of fluid.

Restrictions within the conduits serve several purposes in the presentinvention. In one embodiment, a capillary stop located between thesample holding chamber and first conduit is used to facilitate samplemetering in the holding chamber by preventing displacement of the samplein the holding chamber until sufficient pressure is applied to overcomethe resistance of the capillary stop. In another embodiment, arestriction within the second conduit is used to divert wash fluid alongan alternative pathway towards the waste chamber when the fluid reachesthe constriction. Small holes in the gasket, together with a hydrophobiccoating, are provided to prevent flow from the first conduit to thesecond conduit until sufficient pressure is applied. Features thatcontrol the flow of liquids within and between the conduits of thepresent invention are herein collectively termed “valves.”

One embodiment of the invention provides a single-use cartridge with asample holding chamber connected to a first conduit that contains ananalyte sensor or array of analyte sensors. A second conduit, partlycontaining a fluid, is connected to the first conduit and air segmentscan be introduced into the fluid in the second conduit in order tosegment it. Pump means are provided to displace the sample within thefirst conduit, and this displaces fluid from the second conduit into thefirst conduit. Thus, the sensor or sensors can be contacted first by asample and then by a second fluid.

In another embodiment, the cartridge includes a closeable valve locatedbetween the first conduit and a waste chamber. This embodiment permitsdisplacement of the fluid from the second conduit into the first conduitusing only a single pump means connected to the first conduit. Thisembodiment further permits efficient washing of the conduits of thecartridge of the present invention, which is an important feature of asmall single-use cartridge. In operation, the sample is displaced tocontact the sensors, and is then displaced through the closeable valveinto the waste chamber. Upon wetting, the closeable valve seals theopening to the waste chamber, providing an airtight seal that allowsfluid in the second conduit to be drawn into contact with the sensorsusing only the pump means connected to the first conduit. In thisembodiment, the closeable valve permits the fluid to be displaced inthis manner and prevents air from entering the first conduit from thewaste chamber.

In another embodiment, both a closeable valve and means for introducingsegments into the conduit are provided. This embodiment has manyadvantages, among which is the ability to reciprocate a segmented fluidover the sensor or array of sensors. Thus a first segment or set ofsegments is used to rinse a sensor, and then a fresh segment replaces itfor taking measurements. Only one pump means (that connected to thefirst conduit) is required.

In another embodiment, analyte measurements are performed in a thin-filmof liquid coating an analyte sensor. Such thin-film determinations arepreferably performed amperometrically. This cartridge differs from theforegoing embodiments in having both a closeable valve that is sealedwhen the sample is expelled through the valve, and an air vent withinthe conduits that permits at least one air segment to be subsequentlyintroduced into the measuring fluid, thereby increasing the efficiencywith which the sample is rinsed from the sensor, and further permittingremoval of substantially all the liquid from the sensor prior tomeasurement, and still further permitting segments of fresh liquid to bebrought across the sensor to permit sequential, repetitive measurementsfor improved accuracy and internal checks of reproducibility.

In non-competitive assay embodiments, as discussed above, the analysisscheme for the detection of low concentrations of immunoactive analyterelies on the formation of an enzyme labeledantibody/analyte/surface-bound antibody “sandwich” complex. Theconcentration of analyte in a sample is converted into a proportionalsurface concentration of an enzyme. The enzyme is capable of amplifyingthe analyte's chemical signal by converting a substrate to a detectableproduct. For example, where alkaline phosphatase is the enzyme, a singleenzyme molecule can produce about nine thousand detectable molecules perminute, providing several orders of magnitude improvement in thedetectability of the analyte compared to schemes in which anelectroactive species is attached to the antibody in place of alkalinephosphatase.

In immunosensor embodiments, it is advantageous to contact the sensorfirst with a sample and then with a wash fluid prior to recording aresponse from the sensor. In some specific embodiments, in addition tobeing amended with an IgG-coated microparticle in order to reduceleukocyte interference, the sample is amended with an antibody-enzymeconjugate (signal antibody) that binds to the analyte of interest withinthe sample before the amended sample contacts the sensor. Bindingreactions in the sample produce an analyte/antibody-enzyme complex. Thesensor comprises an immobilized antibody to the analyte, attached closeto an electrode surface. Upon contacting the sensor, theanalyte/antibody-enzyme complex binds to the immobilized antibody nearthe electrode surface. It is advantageous at this point to remove fromthe vicinity of the electrode as much of the unbound antibody-enzymeconjugate as possible to minimize background signal from the sensor. Theenzyme of the antibody-enzyme complex is advantageously capable ofconverting a substrate, provided in the fluid, to produce anelectrochemically active species. This active species is produced closeto the electrode and provides a current from a redox reaction at theelectrode when a suitable potential is applied (amperometric operation).Alternatively, if the electroactive species is an ion, it can bemeasured potentiometrically. In amperometric measurements the potentialmay either be fixed during the measurement, or varied according to apredetermined waveform. For example, a triangular wave can be used tosweep the potential between limits, as is used in the well-knowntechnique of cyclic voltammetry. Alternatively, digital techniques suchas square waves can be used to improve sensitivity in detection of theelectroactive species adjacent to the electrode. From the current orvoltage measurement, the amount or presence of the analyte in the sampleis calculated. These and other analytical electrochemical methods arewell known in the art.

In embodiments in which the cartridge comprises an immunosensor, theimmunosensor is advantageously microfabricated from a base sensor of anunreactive metal such as gold, platinum or iridium, and a porouspermselective layer which is overlaid with a bioactive layer attached toa microparticle, for example latex particles. The microparticles aredispensed onto the porous layer covering the electrode surface, formingan adhered, porous bioactive layer. The bioactive layer has the propertyof binding specifically to the analyte of interest, or of manifesting adetectable change when the analyte is present, and is most preferably animmobilized antibody directed against the analyte.

Referring to the Figures, the cartridge of the present inventioncomprises a cover, an embodiment of which is shown in FIGS. 1 and 2, abase, an embodiment of which is shown in FIG. 4, and a thin-filmadhesive gasket, an embodiment of which is shown in FIG. 3, disposedbetween the base and the cover. Referring to FIG. 1, the cover 1 is madeof a rigid material, preferably plastic, and is capable of repetitivedeformation at flexible hinge regions 5, 9, 10 without cracking Thecover comprises a lid 2, attached to the main body of the cover by aflexible hinge 9. In operation, after introduction of a sample into thesample holding chamber 34, the lid can be secured over the entrance tothe sample entry port 4, preventing sample leakage, and the lid is heldin place by hook 3. The cover further comprises two paddles 6, 7, thatare moveable relative to the body of the cover, and which are attachedto it by flexible hinge regions 5, 10. Additionally in operation, whenoperated upon by a pump means, paddle 6 exerts a force upon an airbladder comprised of cavity 43, which is covered by thin-film gasket 21,to displace fluids within conduits of the cartridge. When operated by asecond pump means, paddle 7 exerts a force upon the gasket 21, which candeform because of slits 22 cut therein. The cartridge is adapted forinsertion into a reading apparatus, and therefore has a plurality ofmechanical and electrical connections for this purpose. In otherembodiments of the invention, manual operation of the cartridge ispossible.

Upon insertion of the cartridge into a reading apparatus, the gaskettransmits pressure onto a fluid-containing foil pack filled withapproximately 130 μL of analysis/wash solution (“fluid”) located incavity 42, rupturing the package upon spike 38, and expelling fluid intoconduit 39, which is connected via a short transecting conduit in thebase to the sensor conduit. The analysis fluid fills the front of theanalysis conduit first pushing fluid onto a small opening in the tapegasket that acts as a capillary stop. Other motions of the analyzermechanism applied to the cartridge are used to inject one or moresegments into the analysis fluid at controlled positions within theanalysis conduit. These segments are used to help wash the sensorsurface and the surrounding conduit with a minimum of fluid.

In some embodiments, the cover further comprises a hole covered by athin pliable film 8. In operation, pressure exerted upon the film expelsone or more air segments into a conduit 20 through a small hole 28 inthe gasket.

Referring to FIG. 2, the lower surface of the base further comprisessecond conduit 11, and first conduit 15. Second conduit 11 includes aconstriction 12, which controls fluid flow by providing resistance tothe flow of a fluid. Optional coatings 13, 14 provide hydrophobicsurfaces, which together with gasket holes 31, 32, control fluid flowbetween second and first conduits 11, 15. A recess 17 in the baseprovides a pathway for air in conduit 34 to pass to conduit 34 throughhole 27 in the gasket.

Referring to FIG. 3, thin-film gasket 21 comprises various holes andslits to facilitate transfer of fluid between conduits within the baseand the cover and to allow the gasket to deform under pressure wherenecessary. Thus, hole 24 permits fluid to flow from conduit 11 intowaste chamber 44; hole 25 comprises a capillary stop between conduits 34and 15; hole 26 permits air to flow between recess 18 and conduit 40;hole 27 provides for air movement between recess 17 and conduit 34; andhole 28 permits fluid to flow from conduit 19 to waste chamber 44 viaoptional closeable valve 41. Holes 30 and 33 permit the plurality ofelectrodes that are housed within cutaways 35 and 37, respectively, tocontact fluid within conduit 15. In a specific embodiment, cutaway 37houses a ground electrode, and/or a counter-reference electrode, andcutaway 35 houses at least one analyte sensor and, optionally, aconductimetric sensor. In FIG. 3, the tape 21 is slit at 22 to allow thetape enclosed by the three cuts 22 to deform when the instrument appliesa downward force to rupture the calibrant pouch within element 42 on thebarb 38. The tape is also cut at 23 and this allows the tape to flexdownwards into element 43 when the instrument provides a downward force,expelling air from chamber 43 and moving the sample fluid throughconduit 15 towards the sensors. Element 29 in FIG. 3 acts as an openingin the tape connecting a region in the cover FIG. 2 with the base FIG.4.

Referring to FIG. 4, conduit 34 is the sample holding chamber thatconnects the sample entry port 4 to first conduit 11 in the assembledcartridge. Cutaway 35 houses the analyte sensor or sensors, or ananalyte responsive surface, together with an optional conductimetricsensor or sensors. Cutaway 37 houses a ground electrode if needed as areturn current path for an electrochemical sensor, and may also house anoptional conductimetric sensor. Cutaway 36 provides a fluid path betweengasket holes 31 and 32 so that fluid can pass between the first andsecond conduits. Recess 42 houses a fluid-containing package, e.g., arupturable pouch, in the assembled cartridge that is pierced by spike 38because of pressure exerted upon paddle 7 upon insertion into a readingapparatus. Fluid from the pierced package flows into the second conduitat 39. An air bladder is comprised of recess 43 which is sealed on itsupper surface by gasket 21. The air bladder is one embodiment of a pumpmeans, and is actuated by pressure applied to paddle 6 which displacesair in conduit 40 and thereby displaces the sample from sample chamber34 into first conduit 15.

The location at which air enters the sample holding chamber (gasket hole27) from the bladder, and the capillary stop 25, together define apredetermined volume of the sample holding chamber. An amount of thesample corresponding to this volume is displaced into the first conduitwhen paddle 6 is depressed. This arrangement is therefore one possibleembodiment of a metering means for delivering a metered amount of anunmetered sample into the conduits of the cartridge.

In certain embodiments of the cartridge, a means for metering a samplesegment is provided in the base plastic part. The segment size iscontrolled by the size of the compartment in the base and the positionof the capillary stop and air pipe holes in the tape gasket. This volumecan be readily varied from 2 to 200 μL. Expansion of this range ofsample sizes is possible within the context of the present invention.

In some embodiments, the fluid is pushed through a pre-analyticalconduit 11 that can be used to amend a reagent (e.g., particles, solublemolecules, or the IgM or fragments thereof) into the sample prior to itspresentation at the sensor conduit 19. Alternatively, the amendingreagent may be located in portion 15, beyond portion 16. Pushing thesample through the pre-analytical conduit also serves to introducetension into the diaphragm pump paddle 7, which improves itsresponsiveness for actuation of fluid displacement.

According to certain embodiments of the invention, metering isadvantageous in some assays if quantification of the analyte isrequired. A waste chamber is provided, 44, for sample and/or fluid thatis expelled from the conduit, to prevent contamination of the outsidesurfaces of the cartridge. A vent connecting the waste chamber to theexternal atmosphere is also provided, 45. One desirable feature of thecartridge is that once a sample is loaded, analysis can be completed andthe cartridge discarded without the operator or others contacting thesample.

Referring now to FIG. 5, a schematic diagram of the features of acartridge and components is provided, wherein 51-57 are portions of theconduits and sample chamber that can optionally be coated with dryreagents to amend a sample or fluid. The sample or fluid is passed atleast once over the dry reagent to dissolve it. Reagents used to amendthe sample may include one or more of the following: antibody-enzymeconjugates (signal antibodies), one or more leukocidal reagents, IgMand/or fragments thereof, IgG and/or fragments thereof, and otherblocking agents that prevent either specific or non-specific bindingreactions among assay compounds, and/or the above-described IgG-coatedmicroparticles for reducing leukocyte interference. A surface coatingthat is not soluble but helps prevent non-specific adsorption of assaycomponents to the inner surfaces of the cartridges can also be provided.

In specific embodiments, a closeable valve is provided between the firstconduit and the waste chamber. In one embodiment, this valve, 58, iscomprised of a dried sponge material that is coated with an impermeablesubstance. In operation, contacting the sponge material with the sampleor a fluid results in swelling of the sponge to fill the cavity 41 (FIG.4), thereby substantially blocking further flow of liquid into the wastechamber 44. Furthermore, the wetted valve also blocks the flow of airbetween the first conduit and the waste chamber, which permits the firstpump means connected to the sample chamber to displace fluid within thesecond conduit, and to displace fluid from the second conduit into thefirst conduit in the following manner.

Referring now to FIG. 6, which illustrates the schematic layout of animmunosensor cartridge, there are provided three pumps, 61-63. Whilethese pumps have been described in terms of specific embodiments, itwill be readily understood that any pumping device capable of performingthe respective functions of pumps 61-63 may be used within the presentinvention. Thus, pump 1, 61, should be capable of displacing the samplefrom the sample holding chamber into the first conduit; pump 2, 62,should be capable of displacing fluid within the second conduit; andpump 3, 63, should be capable of inserting at least one segment into thesecond conduit. Other types of pumps that are envisaged in the presentapplication include, but are not limited to, an air sac contacting apneumatic means whereby pressure is applied to the air sac, a flexiblediaphragm, a piston and cylinder, an electrodynamic pump, and a sonicpump. With reference to pump 3, 63, the term “pump” includes all devicesand methods by which one or more segments are inserted into the secondconduit, such as a pneumatic means for displacing air from an air sac, adry chemical that produces a gas when dissolved, or a plurality ofelectrolysis electrodes operably connected to a current source. In aspecific embodiment, the segment is produced using a mechanical segmentgenerating diaphragm that may have more than one air bladder or chamber.As shown, the well 8 has a single opening which connects the innerdiaphragm pump and the fluid filled conduit into which a segment is tobe injected 20. The diaphragm can be segmented to produce multiplesegments, each injected in a specific location within a fluid filledconduit. In FIG. 6, element 64 indicates the region where theimmunosensor performs the capture reaction to form a sandwich comprisingthe immobilized antibody, the analyte and the signal antibody.

In alternative embodiments, a segment is injected using a passivefeature. A well in the base of the cartridge is sealed by the tapegasket. The tape gasket covering the well has two small holes on eitherend. One hole is open while the other is covered with a filter materialwhich wets upon contact with a fluid. The well is filled with a loosehydrophilic material such as a cellulose fiber filter, paper filter orglass fiber filter. This hydrophilic material draws the liquid into thewell in the base via capillary action, displacing the air that wasformerly in the well. The air is expelled through the opening in thetape gasket creating a segment whose volume is determined by the volumeof the well and the void volume of the loose hydrophilic material. Thefilter used to cover one of the inlets to the well in the base can bechosen to meter the rate at which the fluid fills the well and therebycontrol the rate at which the segment is injected into the conduit inthe cover. This passive feature permits any number of controlledsegments to be injected at specific locations within a fluid path andrequires a minimum of space.

IX. Devices, Kits and Methods for Reducing or Eliminating LeukocyteInterference

Based on the disclosure herein, it is apparent that embodiments of thepresent invention provide a method of reducing or eliminatinginterference from leukocytes in an analyte immunoassay.

Preferred embodiments are directed to the performance of an immunoassayfor a target analyte in a blood sample by contacting a sample with aleukocidal reagent that substantially inhibits activity of leukocytes inthe sample. An immunoassay is also performed on the sample to detect atarget analyte. As described herein, in some embodiments, the leukocidalreagent can be an oxygen depleting reagent and a glucose depletingreagent, e.g., glucose oxidase with or without an electron acceptor. Inother embodiments, the reagent may constitute hexokinase and a source ofATP, e.g. creatine kinase, ADP and creatine phosphate. Anotheralternative is a glucose dehydrogenase, with for example NAD, NADHoxidase and an electron acceptor, or GDH-PQQ. In other embodiments, anoxygen depleting reagent, e.g. dithionite, glucose oxidase (with orwithout added glucose) and ascorbate oxidase can be used.

The present invention further constitutes embodiments where theleukocidal reagent is a mitochondrial electron transport inhibitorand/or a mitochondrial membrane uncoupling agent, e.g. cyanide,antimycin, rotenone, malonate, carbonyl cyanidep-[trifluoromethoxyl]-phenyl-hydrozone, 2,4-dinitrophenol andoligomycin. In additional embodiments, the reagent may be an amphipathicreagent, e.g. a saponin, a leukocidin and a mucopolysaccharide. Anotheralternative is the use of a monoclonal antibody AHN-1, which inhibitsphagocytosis by human neutrophils, see Skubitz et al, Blood 65, 333-339,(1985), which is herein incorporated by reference.

The present invention is equally applicable to both sandwich andcompetitive immunoassays. In sandwich assay embodiments, the samplecontacts an immunosensor with an immobilized first antibody to thetarget analyte, and a labeled second antibody to said target analyte. Incompetitive assay embodiments, the sample contacts an immunosensorcomprising an immobilized first antibody to said target analyte, and alabeled target analyte that competes for binding with the targetanalyte. Typical analytes include, but are not limited to TnI, TnT, BNP,NTproBNP, proBNP, HCG, TSH, NGAL, digoxin, theophylline and phenytoinand the like.

In some embodiments of the invention, the sample, e.g., whole bloodsample, is first collected and then amended by dissolving a dry reagentcomprising one or more leukocidal reagents and optionally sacrificialbeads into the sample. In certain embodiments, sufficient sacrificialbeads are utilized to provide an excess of beads with respect toleukocytes in a blood sample. This yields a sample with a dissolvedsacrificial bead concentration of at least 5 micrograms per microliterof sample, e.g., at least 10 micrograms per microliter of sample, or atleast 15 micrograms per microliter of sample, which is sufficient tosubstantially engage any leukocytes in the sample. In terms of ranges,the dry reagent preferably dissolves into the sample to give asacrificial bead concentration of from about 5 micrograms to about 40micrograms beads per microliter of sample, preferably from about 10 toabout 20 micrograms beads per microliter of sample. Depending on thesize of the beads, this corresponds to at least about 10⁴ beads permicroliter of sample, at least about 10⁵ beads per microliter of sample,or approximately from about 10⁵ to about 10⁶ beads per microliter ofsample. Thus, in some preferred embodiments, in addition to theleukocidal reagent, the sacrificial beads are present in an amountsufficient to provide a dissolved sacrificial bead concentration of atleast 10⁴ beads per microliter of sample, e.g., at least about 10⁵ beadsper microliter of sample, or from about 10⁵ to about 10⁶ beads permicroliter of sample. Once this step is completed, it is possible toperform an immunoassay, e.g., an electrochemical immunoassay, on theamended sample to determine the concentration of an analyte.

The dissolution of the dry reagents and the sandwich formation step canoccur concurrently or in a stepwise manner. Embodiments of the method ofthe present invention are directed mainly to analytes that arecardiovascular markers, e.g., TnI, TnT, CKMB, myoglobin, BNP, NT-proBNP,and proBNP, but can also be used for other markers such as, for example,beta-HCG, TSH, myeloperoxidase, myoglobin, D-dimer, CRP, NGAL and PSA.To ensure that the majority of the leukocytes are sequestered before thedetection step, it is preferable that the sample amendment step is for aselected predetermined period in the range of about 1 minute to about 30minutes.

In preferred embodiments, the method is performed in a cartridgecomprising an immunosensor, a conduit, a sample entry port and a sampleholding chamber, where at least a portion of at least one of theseelements is coated with the dry reagent. Note that the dry reagent caninclude one or more of: a leukocidal reagent, sacrificial beads forreducing leukocyte interference, buffer, salt, surfactant, stabilizingagent, a simple carbohydrate, a complex carbohydrate and variouscombinations. In addition, the dry reagent can also include anenzyme-labeled antibody (signal antibody) to the analyte.

If sacrificial beads are employed, as suggested above, in addition to orinstead of coating the sacrificial beads using whole IgG molecules,where the individual monomers are formed from an Fc region attached to aF(ab′)2 region, which in turn comprises two Fab regions, it is alsopossible to use fragments of IgG. IgG fragmentation can be achievedvariously using combinations of disulphide bond reduction (—S—S— to —SHHS—) and enzymatic pepsin or papain digestion, to create somecombination of F(ab′)2 fragments, Fab fragments, and/or Fc fragments.These fragments can be separated for use separately by chromatography,or used in combination. For example, where the blocking site is on theFc fragment, this can be used instead of the whole IgG molecule. Thesame applies to the Fab fragment and the F(ab′)2 fragment.

In the actual assay step, in preferred embodiments, once the sandwich isformed between the immobilized and signal antibodies, the sample mediumis subsequently washed to a waste chamber, followed by exposing thesandwich to a substrate capable of reacting with an enzyme to form aproduct capable of electrochemical detection. The preferred format is anelectrochemical enzyme-linked immunosorbent assay.

Preferably, the device is one that performs an immunoassay of an analytein a sample, e.g., blood sample, with reduced interference fromleukocytes. The device is a housing with an electrochemicalimmunosensor, a conduit and a sample entry port, where the conduitpermits the sample to pass from the entry port to the immunosensor in acontrolled manner.

In one aspect, samples containing an analyte of interest may be amendedwith one or more leukocidal reagents that reduce or eliminate theactivity of leukocytes, as described above, and the resulting amendedsample may be analyzed in an immunoassay to determine analyte contentand/or concentration without significant leukocyte interference. Inpreferred embodiments, the impaired activity is phagocytosis. Withregard to various preferred embodiments of the invention, suchembodiments include modifying whole blood samples with glucose oxidaseand glucose to give respective sample concentrations above about 3 IUper mL and 500 mg/dL, with or without saponin at about 0.5 mg/mL, andwith or without opsonized decoy beads as described herein.

In another aspect, in addition to amending the sample with the one ormore leukocidal reagents, at least a portion of the housing may becoated with a dry reagent which can comprise non-human IgG-coatedsacrificial beads. The important feature is that the dry reagent iscapable of dissolving into the sample and engaging leukocytes in bindingand preferably phagocytosis. This is generally sufficient to sequesterpotentially interfering leukocytes in the sample.

In a preferred embodiment, the device also comprises an immuno-referencesensor. The immunosensor is preferably directed to detect acardiovascular marker, e.g., analytes such as TnI, TnT, CKMB, myoglobin,BNP, NT-proBNP, and proBNP. The system in which the device operatesgenerally allows the sample to remain in contact with the reagent for apredetermined period, e.g., from 1 to 30 minutes. Preferably the deviceis a single-use cartridge, e.g., filled with a single sample, used oncefor the test and then discarded. Generally, the device includes a washfluid capable of washing the sample to a waste chamber, and a substratecapable of reacting with the enzyme sandwich at the immunosensor to forma product suitable for electrochemical detection.

More broadly the invention relates to reducing interference fromleukocytes in an analyte immunoassay for any biological sample whereleukocytes are generally present. Furthermore, performing an immunoassayon the amended sample to determine the concentration of the selectedanalyte can be based on various techniques including electrochemicalones, e.g., amperometric and potentiometric, and also optical ones,e.g., absorbance, fluorescence and luminescence.

Various embodiments of the present invention are directed to a kit forperforming an immunoassay for a target analyte in a whole blood samplecomprising a leukocidal reagent wherein said reagent substantiallyinhibits activity of leukocytes in a sample, and immunoassay reagentsfor detecting a target analyte. Other embodiments of the invention aredirected to a kit for performing an immunoassay for an analyte suspectedof being present in a blood sample where the kit comprises one or moreleukocidal reagents, sacrificial beads opsonized to leukocytes, animmobilized first antibody to the analyte and a second labeled antibodyto the analyte. In addition a current immunoassay format known in theart may be modified to include the sacrificial beads, for example byadding the beads in a sample pre-treatment step. This pretreatment maybe accomplished by incorporating the sacrificial beads in a bloodcollection device, in a separate vessel, or may take place in theanalytical (immunoassay) device itself by incorporation of thesacrificial beads in the test cycle of the device.

While the present invention has been described in the context of a BNPtest cartridge, it is equally applicable to any immunoassay whereleukocytes are present and can be the cause of interference. The methodor kit is not limited to BNP but can be adapted to any immunoassay,including but not limited to proBNP, NTproBNP, cTnI, TnT, HCG, TSH, PSA,D-dimer, CRP, myoglobin, NGAL, CKMB and myeloperoxidase. Furthermore themethod and kit is applicable to assays where the sacrificial beads, ifemployed, are coated with any non-human IgG or a fragment thereof,including murine, caprine, bovine and lupine, and alternativelysacrificial beads coated with an activated human IgG or fragmentthereof. The sacrificial beads may comprise substrate beads coated witha material or fragment thereof selected from a protein, a bacterium, avirus and a xenobiotic, or may be afforded by dormant or otherwisestabilized bacterial cells, spores or fragment thereof, e.g., E. coli,optionally without substrate beads.

While certain assays described above use an immobilized first antibodyattached to an assay bead which is in turn attached to a porous layerwith an underlying electrode, the first antibody may be immobilizeddirectly onto an electrode or any other surface, or immobilized on asoluble bead.

The kit or method of the present invention can comprise a second labeledantibody that is in the form of a dissolvable dry reagent. In someembodiments, the dissolvable dry reagent includes one or more leukocidalreagents and optionally opsonized sacrificial beads as part of thedissolvable dry reagent, or where the various components are in separatedry reagent locations. Note that both the immobilized and labeledantibodies can be monoclonal, polyclonal, fragments thereof andcombinations thereof. In addition, the second antibody can be labeledwith various labels including a radiolabel, enzyme, chromophore,flurophore, chemiluminescent species and other known in the immunoassayart. Where the second antibody is labeled with an enzyme, it ispreferably ALP, horseradish peroxidase, or glucose oxidase.

The kit or method is applicable to any sample containing leukocytes,e.g., whole blood, and can be a blood sample amended with ananticoagulant, e.g., EDTA, heparin, fluoride, citrate and the like.

Where the method or kit is used to perform a non-competitive immunoassaythere may be a sequence of mixing steps including: (i) mixing a bloodsample suspected of containing an analyte with reagent (including one ormore leukocidal reagents) and optionally opsonized sacrificial beads;(ii) mixing the blood sample with an immobilized first antibody to theanalyte and forming a complex between the immobilized antibody and saidanalyte; and (iii) mixing the blood sample with a labeled secondantibody to form a complex with said analyte and said immobilizedantibody. Note that these mixing steps can be performed at the same timeor in an ordered sequence. For example, steps (ii) and (iii) may occurat the same time, or steps (i) and (iii) may be performed before step(ii). In the final step there is a determination of the amount ofcomplex formed between the immobilized first antibody, the analyte andthe labeled second antibody.

Embodiments of the method of the present invention are directed tosubstantially ameliorating white blood cell accumulation on an analyteimmunosensor. In some embodiments, the immunosensor is made from ananti-body-based reagent and/or antibody-coated assay beads to theanalyte. After mixing a sample suspected of containing an analyte withthe one or more leukocidal reagents and optionally opsonized sacrificialbeads to form an amended sample where white blood cells in the samplepreferentially are ameliorated by the one or more leukocidal reagents orseek to preferentially phagocytose the sacrificial beads, if present,the amended sample is then contacted with the immunosensor. As a resultthere is minimal accumulation of white cells, and a reliable assay maybe achieved.

The present method is directed to performing an immunoassay for ananalyte suspected of being present in a blood sample comprising: mixinga blood sample suspected of containing an analyte with one or moreleukocidal reagents and optionally with an excess of opsonizedsacrificial beads to form an amended sample wherein white blood cells inthe sample are ameliorated with the leukocidal reagents and/orpreferentially seek to phagocytose the sacrificial beads (if present);contacting the amended sample with an immunosensor comprisingantibody-coated beads to the analyte immobilized on an electrode;forming a sandwich between said antibody-coated bead, said analyte and asecond labeled antibody; washing said blood sample from saidimmunosensor; and determining the amount of said sandwiched label withsaid immunosensor and relating the amount of said label to theconcentration of the analyte in the sample.

The present method is also directed to performing an immunoassay for ananalyte suspected of being present in a blood sample comprising: mixinga blood sample suspected of containing an analyte with one or moreleukocidal reagents and optionally with an excess of opsonizedsacrificial beads to form an amended sample wherein white blood cells inthe sample are ameliorated with the one or more leukocidal reagents and,if present, preferentially seek to phagocytose the sacrificial beads;contacting the amended sample with the reagent and/or beads to theanalyte; forming a sandwich between said reagent and/or beads, saidanalyte and a second labeled antibody; washing said blood sample fromsaid reagent and/or beads; and determining the amount of said sandwichedlabel and relating the amount of said label to the concentration of theanalyte in the sample.

The present invention is also directed to a test cartridge forperforming an immunoassay for an analyte suspected of being present in ablood sample comprising: an immunosensor in a conduit wherein saidimmunosensor having an immobilized first antibody to the analyte. Insome embodiments, the conduit preferably has a dry reagent coating orseparate coatings comprising one or more leukocidal reagents andoptionally sacrificial beads opsonized to leukocytes and a secondlabeled antibody to said analyte. In operation, the dry reagentdissolves into said blood sample.

EXAMPLES

The present invention will be better understood in view of the followingnon-limiting Examples.

Example 1

FIG. 7 illustrates the principle of an amperometric immunoassayaccording to specific embodiments of U.S. application Ser. No.12/411,325, referenced above and incorporated herein by reference in itsentirety for determining the presence and amount of troponin I (TnI), amarker of cardiac necrosis. A blood sample was introduced into thesample holding chamber of a cartridge and was amended by dissolution ofa dry reagent coated into the sample holding chamber. The dry reagentincludes IgM 77, which upon dissolution into the sample selectivelybinds to complementary heterophile antibodies 78 that may be containedin the sample. As shown, the dry reagent also comprises IgG 79, whichalso selectively binds to complementary antibodies 78 after dissolutioninto the sample.

FIG. 10 illustrates the principle of an amperometric immunoassayaccording to U.S. patent application Ser. Nos. 12/620,179 and12/620,230, for determining the presence and amount of a marker ofcardiac function, e.g., BNP and TnI, and employing opsonized sacrificialbeads for reducing leukocyte interference. A blood sample was introducedinto the sample holding chamber of a cartridge and was amended bydissolution of a dry reagent coated into the sample holding chamber. Thedry reagent includes sacrificial beads 102, which upon dissolution intothe sample selectively bind to leukocytes 103 that may be contained inthe sample. Note that the dry reagent may also and preferably comprisesIgM and IgG as described for FIG. 7.

In addition, FIGS. 7 and 10 show a conjugate molecule comprisingalkaline phosphatase enzyme (AP) covalently attached to a signalantibody 71, e.g., polyclonal anti-troponin I antibody, also wasdissolved into the sample. This conjugate specifically binds to theanalyte 70, e.g., TnI or BNP, in the blood sample producing a complexmade up of analyte bound to the AP conjugate. In a capture step, thiscomplex binds to the capture antibody 72 (immobilized antibody) attachedon, or close to, the immunosensor. The sensor chip has a conductivitysensor, which is used to monitor when the sample reaches the sensorchip. The time of arrival of the fluid can be used to detect leakswithin the cartridge, e.g., a delay in arrival signals a leak. In someembodiments, the position of the sample segment within the sensorconduit can be actively controlled using the edge of the fluid as aposition marker. As the sample/air interface crosses the conductivitysensor, a precise signal is generated which can be used as a fluidmarker from which controlled fluid excursions can be executed. The fluidsegment is preferentially oscillated edge-to-edge over the sensor inorder to present the entire sample to the immunosensor surface. A secondreagent can be introduced in the sensor conduit beyond the sensor chip,which becomes homogenously distributed during the fluid oscillations.

The sensor chip contains a capture region or regions coated withantibodies for the analyte of interest. These capture regions aredefined by a hydrophobic ring of polyimide or anotherphotolithographically produced layer. A microdroplet or severalmicrodroplets (approximately 5 to 40 nanoliters in size) containingantibodies in some form, for example bound to latex microspheres, isdispensed on the surface of the sensor or on a permselective layer onthe sensor. The photodefined ring contains this aqueous droplet allowingthe antibody coated region to be localized to a precision of a fewmicrons. The capture region can be made from 0.03 to roughly 2 squaremill imeters in size. The upper end of this size is limited by the sizeof the conduit and sensor in present embodiments, and is not alimitation of the invention.

Thus, the gold electrode 74 is coated with a biolayer 73, e.g., acovalently attached anti-troponin I antibody, to which the TnI/AP-aTnIcomplex binds. AP is thereby immobilized close to the electrode inproportion to the amount of analyte initially present in the sample. Inaddition to specific binding, the enzyme-antibody conjugate may bindnon-specifically to the sensor. Non-specific binding provides abackground signal from the sensor that is undesirable and preferably isminimized. As described above, the rinsing protocols, and in particularthe use of segmented fluid to rinse the sensor, provide efficient meansto minimize this background signal. In a second step subsequent to therinsing step, a substrate 75 that is hydrolyzed by, for example,alkaline phosphatase to produce an electroactive product 76 is presentedto the sensor. In specific embodiments the substrate is comprised of aphosphorylated ferrocene or p-aminophenol. The amperometric electrode iseither poised at a fixed electrochemical potential sufficient to oxidizeor reduce a product of the hydrolyzed substrate but not the substratedirectly, or the potential is swept one or more times through anappropriate range. Optionally, a second electrode may be coated with alayer where the complex of analyte/AP anti-analyte, e.g., TnI/AP-aTnI,is made during manufacture to act as a reference sensor or calibrationmeans for the measurement.

In the present example, the sensor comprises two amperometric electrodeswhich are used to detect the enzymatically produced 4-aminophenol fromthe reaction of 4-aminophenylphosphate with the enzyme label alkalinephosphatase. The electrodes are preferably produced from gold surfacescoated with a photodefined layer of polyimide. Regularly spaced openingsin the insulating polyimide layer define a grid of small gold electrodesat which the 4-aminophenol is oxidized in a two electron per moleculereaction. Sensor electrodes further comprise a biolayer, while referenceelectrodes can be constructed, for example, from gold electrodes lackinga biolayer, or from silver electrodes, or other suitable material.Different biolayers can provide each electrode with the ability to sensea different analyte.

Substrates, such as p-aminophenol species, can be chosen such that theE(1/2) of the substrate and product differ substantially. Preferably,the voltammetric half-wave potential E(1/2) of the substrate issubstantially higher (more positive) than that of the product. When thecondition is met, the product can be selectively electrochemicallymeasured in the presence of the substrate.

The size and spacing of the electrode play an important role indetermining the sensitivity and background signal. Important parametersin the grid include the percentage of exposed metal and the spacingbetween the active electrodes. The position of the electrode can bedirectly underneath the antibody capture region or offset from thecapture region by a controlled distance. The actual amperometric signalof the electrodes depends on the positioning of the sensors relative tothe antibody capture site and the motion of the fluid during theanalysis. A current at the electrode is recorded that depends upon theamount of electroactive product in the vicinity of the sensor.

The detection of alkaline phosphatase activity in this example relies ona measurement of the 4-aminophenol oxidation current. This is achievedat a potential of about +60 mV versus the Ag/AgCl ground chip. The exactform of detection used depends on the sensor configuration. In oneversion of the sensor, the array of gold microelectrodes is locateddirectly beneath the antibody capture region. When the analysis fluid ispulled over this sensor, enzyme located on the capture site converts the4-aminophenylphosphate to 4-aminophenol in an enzyme limited reaction.The concentration of the 4-aminophenylphosphate is selected to be inexcess, e.g., 10 times the Km value. The analysis solution is 0.1 M indiethanolamine, 1.0 M NaCl, buffered to a pH of 9.8. In additionalembodiments, the analysis solution contains 0.5 mM MgCl₂, which is acofactor for the enzyme. Alternatively, a carbonate buffer has thedesired properties.

In another electrode geometry embodiment, the electrode is located a fewhundred microns away from the capture region. When a fresh segment ofanalysis fluid is pulled over the capture region, the enzyme productbuilds with no loss due to electrode reactions. After time, the solutionis slowly pulled from the capture region over the detector electroderesulting in a current spike from which the enzyme activity can bedetermined.

An important consideration in the sensitive detection of alkalinephosphatase activity is the non-4-aminophenol current associated withbackground oxidations and reductions occurring at the gold sensor. Goldsensors tend to give significant oxidation currents in basic buffers atthese potentials. The background current is largely dependent on thebuffer concentration, the area of the gold electrode (exposed area),surface pretreatments and the nature of the buffer used. Diethanolamineis a particularly good activating buffer for alkaline phosphatase. Atmolar concentrations, the enzymatic rate is increased by about threetimes over a non-activating buffer such as carbonate.

In alternative embodiments, the enzyme conjugated to an antibody orother analyte-binding molecule is urease, and the substrate is urea.Ammonium ions produced by the hydrolysis of urea are detected in thisembodiment by the use of an ammonium sensitive electrode.Ammonium-specific electrodes are well-known to those of skill in theart. For example, a suitable microfabricated ammonium ion-selectiveelectrode is disclosed in U.S. Pat. No. 5,200,051, incorporated hereinby reference. Other enzymes that react with a substrate to produce anion are known in the art, as are other ion sensors for use therewith.For example, phosphate produced from an alkaline phosphatase substratecan be detected at a phosphate ion-selective electrode.

FIG. 8 illustrates the construction of an embodiment of amicrofabricated immunosensor. Preferably a planar non-conductingsubstrate 80 is provided onto which is deposited a conducting layer 81by conventional means or microfabrication known to those of skill in theart. The conducting material is preferably a noble metal such as gold orplatinum, although other unreactive metals such as iridium may also beused, as may non-metallic electrodes of graphite, conductive polymer, orother materials. An electrical connection 82 is also provided. Abiolayer 83 is deposited onto at least a portion of the electrode. Inthe present disclosure, a “biolayer” refers to a porous layer comprisingon its surface a sufficient amount of a molecule 84 that can either bindto an analyte of interest, or respond to the presence of such analyte byproducing a change that is capable of measurement. Optionally, apermselective screening layer may be interposed between the electrodeand the biolayer to screen electrochemical interferents, as described inU.S. Pat. No. 5,200,051, referenced above.

In specific embodiments, a biolayer is constructed from latex beads ofspecific diameter in the range of about 0.001 to 50 microns. The beadsare modified by covalent attachment of any suitable molecule consistentwith the above definition of a biolayer. Many methods of attachmentexist in the art, including providing amine reactiveN-hydroxysuccinimide ester groups for the facile coupling of lysine orN-terminal amine groups of proteins. In specific embodiments, thebiomolecule is chosen from among ionophores, cofactors, polypeptides,proteins, glycopeptides, enzymes, immunoglobulins, antibodies, antigens,lectins, neurochemical receptors, oligonucleotides, polynucleotides,DNA, RNA, or suitable mixtures. In more specific embodiments, thebiomolecule is an antibody selected to bind one or more of humanchorionic gonadotrophin, troponin I, troponin T, troponin C, a troponincomplex, creatine kinase, creatine kinase subunit M, creatine kinasesubunit B, myoglobin, myosin light chain, or modified fragments ofthese. Such modified fragments are generated by oxidation, reduction,deletion, addition or modification of at least one amino acid, includingchemical modification with a natural moiety or with a synthetic moiety.Preferably, the biomolecule binds to the analyte specifically and has anaffinity constant for binding analyte ligand of about 10⁻⁷ to 10 ⁻¹⁵ M.

In one embodiment, the biolayer, comprising beads having surfaces thatare covalently modified by a suitable molecule, is affixed to the sensorby the following method. A microdispensing needle is used to depositonto the sensor surface a small droplet, preferably about 20 nL, of asuspension of modified beads. The droplet is permitted to dry, whichresults in a coating of the beads on the surface that resistsdisplacement during use.

In addition to immunosensors in which the biolayer is in a fixedposition relative to an amperometric sensor, the present invention alsoenvisages embodiments in which the biolayer is coated upon particles,e.g., assay beads, that are mobile. The cartridge can contain mobilemicroparticles capable of interacting with an analyte, for examplemagnetic particles that are localized to an amperometric electrodesubsequent to a capture step, whereby magnetic forces are used toconcentrate the particles at the electrode for measurement. Oneadvantage of mobile microparticles in the present invention is thattheir motion in the sample or fluid accelerates binding reactions,making the capture step of the assay faster. For embodiments usingnon-magnetic mobile microparticles, a porous filter is used to trap thebeads at the electrode. Note that with respect to the sacrificial beads,where the assay beads are magnetic, the sacrificial beads are preferablynon-magnetic and sequestered separately. In addition, where the assaybeads are non-magnetic, the sacrificial beads are preferably magneticand sequestered separately.

Referring now to FIG. 9, there is illustrated a mask design for severalelectrodes upon a single substrate. By masking and etching techniques,independent electrodes and leads can be deposited. Thus, a plurality ofimmunosensors, 94 and 96, and conductimetric sensors, 90 and 92, areprovided in a compact area at low cost, together with their respectiveconnecting pads, 91, 93, 95, and 97, for effecting electrical connectionto the reading apparatus. In principle, a very large array of sensorscan be assembled in this way, each sensitive to a different analyte oracting as a control sensor or reference immunosensor.

In specific embodiments of the invention, immunosensors may be preparedas follows. Silicon wafers are thermally oxidized to form an insulatingoxide layer approximately 1 micron thick. A titanium/tungsten layer issputtered onto the oxide layer to a preferable thickness of between 100to 1000 Angstroms, followed by a layer of gold that is most preferably800 Angstroms thick. Next, a photoresist is spun onto the wafer and isdried and baked appropriately. The surface is then exposed using acontact mask, such as a mask corresponding to that illustrated in FIG.9. The latent image is developed, and the wafer is exposed to agold-etchant. The patterned gold layer is coated with a photodefinablepolyimide, suitably baked, exposed using a contact mask, developed,cleaned in an O₂ plasma, and preferably imidized at 350° C. for 5 hours.An optional metallization of the back side of the wafer may be performedto act as a resistive heating element, where the immunosensor is to beused in a thermostatted format. The surface is then printed withantibody-coated particles. Droplets, preferably of about 20 nL volumeand containing 1% solid content in deionized water, are deposited ontothe sensor region and are dried in place by air drying. Optionally, anantibody stabilization reagent (supplied by SurModica Corporation, EdenPrairie, Minn., USA or Applied Enzyme Technology Ltd, Pontypool, UK) isovercoated onto the sensor.

Drying the particles causes them to adhere to the surface in a mannerthat prevents dissolution in either sample or fluid containing asubstrate. This method provides a reliable and reproducibleimmobilization process suitable for manufacturing sensor chips in highvolume.

Example 2

With respect to the method of use of a cartridge, in one embodiment, anunmetered fluid sample is introduced into sample holding chamber 34 of acartridge, through sample entry port 4. Capillary stop 25 preventspassage of the sample into conduit 15 at this stage, and holding chamber34 is filled with the sample. Lid 2 or element 200 is closed to preventleakage of the sample from the cartridge. The cartridge is then insertedinto a reading apparatus, such as that disclosed in U.S. Pat. No.5,821,399 to Zelin, which is hereby incorporated by reference. Insertionof the cartridge into a reading apparatus activates the mechanism, whichpunctures a fluid-containing package located at 42 when the package ispressed against spike 38. Fluid is thereby expelled into the secondconduit, arriving in sequence at 39, 20, 12 and 11. The constriction at12 prevents further movement of fluid because residual hydrostaticpressure is dissipated by the flow of fluid via second conduit portion11 into the waste chamber 44. In a second step, operation of a pumpmeans applies pressure to air bladder 43, forcing air through conduit40, through cutaways 17 and 18, and into conduit 34 at a predeterminedlocation 27. Capillary stop 25 and location 27 delimit a metered portionof the original sample. While the sample is within sample holdingchamber 34, it is amended with the dry reagent coating comprising one ormore leukocidal reagents and optionally sacrificial beads and any otherdesired materials on the inner surface of the chamber. The meteredportion of the sample is then expelled through the capillary stop by airpressure produced within air bladder 43. The sample passes into conduit15 and into contact with the analyte sensor or sensors located withincutaway 35.

In embodiments employing an immunosensor located within cutout 35, thesample is amended prior to arriving at the sensor by, for example, anenzyme-antibody conjugate (signal antibody). To promote efficientbinding of the analyte to the sensor, the sample containing the analyteis optionally passed repeatedly over the sensor in an oscillatorymotion. Preferably, an oscillation frequency of between about 0.2 and 2Hz is used, most preferably 0.7 Hz. Thus, the signal enzyme associatedwith the signal antibody is brought into close proximity to theamperometric electrode surface in proportion to the amount of analytepresent in the sample.

Once an opportunity for the analyte/enzyme-antibody conjugate complex tobind to the immunosensor has been provided, the sample is ejected byfurther pressure applied to air bladder 43, and the sample passes towaste chamber 44. A wash step next removes non-specifically boundenzyme-conjugate and sacrificial beads from the sensor chamber. Fluid inthe second conduit is moved by a pump means 43, into contact with thesensors. The analysis fluid is pulled slowly until the first air segmentis detected at a conductivity sensor.

The air segment or segments can be produced within a conduit by anysuitable means, including but not limited to: (1) passive means, asshown in FIG. 14 and described below; (2) active means including atransient lowering of the pressure within a conduit using a pump wherebyair is drawn into the conduit through a flap or valve; or (3) bydissolving a compound pre-positioned within a conduit that liberates agas upon contacting fluid in the conduit, where such compound mayinclude a carbonate, bicarbonate or the like. This segment is extremelyeffective at clearing the sample-contaminated fluid from conduit 15. Theefficiency of the rinsing of the sensor region is greatly enhanced bythe introduction of one or more air segments into the second conduit asdescribed. The leading and/or trailing edges of air segments are passedone or more times over the sensors to rinse and re-suspend extraneousmaterial that may have been deposited from the sample. Extraneousmaterial includes any material other than specifically bound analyte oranalyte/antibody-enzyme conjugate complex. However, it is an object ofthe invention that the rinsing is not sufficiently protracted orvigorous as to promote dissociation of specifically bound analyte oranalyte/antibody-enzyme conjugate complex from the sensor.

Another advantage of introducing air segments into the fluid is tosegment the fluid. For example, after a first segment of the fluid isused to rinse a sensor, a second segment is then placed over the sensorwith minimal mixing of the two segments. This feature further reducesbackground signal from the sensor by more efficiently removing unboundantibody-enzyme conjugate. After the front edge washing, the analysisfluid is pulled slowly until the first air segment is detected at aconductivity sensor. This segment is extremely effective at clearing thesample-contaminated fluid which was mixed in with the first analysisfluid sample. For measurement, a new portion of fluid is placed over thesensors, and the current or potential, as appropriate to the mode ofoperation, is recorded as a function of time.

Example 3

Referring now to FIG. 15, there is shown a top view of an immunosensorcartridge. Cartridge 150 comprises a base and a top portion, preferablyconstructed of a plastic. The two portions are connected by a thin,adhesive gasket or thin pliable film. As in previous embodiments, theassembled cartridge comprises a sample holding chamber 151 into which asample containing an analyte of interest is introduced via a sampleinlet 167. A metered portion of the sample is delivered to the sensorchip 153, via the sample conduit 154 (first conduit) as before by thecombined action of a capillary stop 152, preferably formed by a 0.012inch (0.3 mm) laser cut hole in the gasket or film that connects the twoportions of the cartridge, and an entry point 155 located at apredetermined point within the sample holding chamber whereby airintroduced by the action of a pump means, such as a paddle pushing upona sample diaphragm 156. After contacting the sensor to permit binding tooccur, the sample is moved to vent 157, which contains a wickingmaterial that absorbs the sample and thereby seals the vent closed tothe further passage of liquid or air. The wicking material is preferablya cotton fiber material, a cellulose material, or other hydrophilicmaterial having pores. It is important in the present application thatthe material is sufficiently absorbent (i.e., possesses sufficientwicking speed) that the valve closes within a time period that iscommensurate with the subsequent withdrawal of the sample diaphragmactuating means described below, so that sample is not subsequentlydrawn back into the region of the sensor chip.

As in the specific embodiment shown, there is provided a wash conduit(second conduit) 158, connected at one end to a vent 159 and at theother end to the sample conduit at a point 160 of the sample conduitthat is located between vent 157 and sensor chip 153. Upon insertion ofthe cartridge into a reading apparatus, a fluid is introduced intoconduit 158. Preferably, the fluid is present initially within a foilpouch 161 that is punctured by a pin when an actuating means appliespressure upon the pouch. There is also provided a short conduit 162 thatconnects the fluid to conduit 154 via a small opening in the gasket 163.A second capillary stop initially prevents the fluid from reachingcapillary stop 160, so that the fluid is retained within conduit 158.

After vent 157 has closed, the pump is actuated, creating a loweredpressure within conduit 154. Air vent 164, preferably comprising a smallflap cut in the gasket or a membrane that vibrates to provide anintermittent air stream, provides a means for air to enter conduit 158via a second vent 165. The second vent 165 preferably also containswicking material capable of closing the vent if wetted, which permitssubsequent depression of sample diaphragm 156 to close vent 165, ifrequired. Simultaneously with the actuation of sample diaphragm 156,fluid is drawn from conduit 158, through capillary stop 160, intoconduit 154. Because the flow of fluid is interrupted by air enteringvent 164, at least one air segment (a segment or stream of segments) isintroduced.

Further withdrawal of sample diaphragm 156 draws the liquid containingat least one air segment back across the sensing surface of sensor chip153. The presence of air-liquid boundaries within the liquid enhancesthe rinsing of the sensor chip surface to remove remaining sample.Preferably, the movement of the sample diaphragm 156 is controlled inconjunction with signals received from the conductivity electrodeshoused within the sensor chip adjacent to the analyte sensors. In thisway, the presence of liquid over the sensor is detected, and multiplereadings can be performed by movement of the fluid in discrete steps.

It is advantageous in this embodiment to perform analyte measurementswhen only a thin film of fluid coats the sensors, ground chip 165, and acontiguous portion of the wall of conduit 154 between the sensors andground electrode. A suitable film is obtained by withdrawing fluid byoperation of the sample diaphragm 156, until the conductimetric sensorlocated next to the sensor indicates that bulk fluid is no longerpresent in that region of conduit 154. It has been found thatmeasurement can be performed at very low (nA) currents, the potentialdrop that results from increased resistance of a thin film betweenground chip and sensor chip (compared to bulk fluid), is notsignificant.

The ground chip 165 is preferably silver/silver chloride. It isadvantageous, to avoid air segments, which easily form upon therelatively hydrophobic silver chloride surface, to pattern the groundchip as small regions of silver/silver chloride interspersed with morehydrophilic regions, such as a surface of silicon dioxide. Thus, apreferred ground electrode configuration comprises an array ofsilver/silver chloride squares densely arranged and interspersed withsilicon dioxide. There is a further advantage in the avoidance ofunintentional segments if the regions of silver/silver chloride aresomewhat recessed.

Referring now to FIG. 16, there is shown a schematic view of thefluidics of the preferred embodiment of an immunosensor cartridge.Regions R1-R7 represent specific regions of the conduits associated withspecific operational functions. Thus R1 represents the sample holdingchamber; R2 the sample conduit whereby a metered portion of the sampleis transferred to the capture region, and in which the sample isoptionally amended with a substance coated upon the walls of theconduit, e.g., one or more leukocidal reagents and optionally opsonizedsacrificial beads; R3 represents the capture region, which houses theconductimetric and analyte sensors; R4 and R5 represent portions of thefirst conduit that are optionally used for further amendment of fluidswith substances coated onto the conduit wall, whereby more complex assayschemes are achieved; R6 represents the portion of the second conduitinto which fluid is introduced upon insertion of the cartridge into areading apparatus; R7 comprises a portion of the conduit located betweencapillary stops 160 and 166, in which further amendment can occur; andR8 represents the portion of conduit 154 located between point 160 andvent 157, and which can further be used to amend liquids containedwithin.

Example 4

With regard to the coordination of fluidics and analyte measurements,during the analysis sequence, a user places a sample into the cartridge,places the cartridge into the analyzer and in from 1 to 20 minutes, aquantitative measurement of one or more analytes is performed. Anon-limiting example of a sequence of events that occur during theanalysis is as follows:

(1) A 25 to 50 μL sample is introduced in the sample inlet 167 and fillsto a capillary stop 151 formed by a 0.012 inch (0.3 mm) laser cut holein the adhesive tape holding the cover and base components together. Oneor more dry reagent coatings comprising one or more leukocidal reagentsand optionally opsonized sacrificial beads plus optionally othermaterials for ameliorating interference and preferably a signal antibodyare dissolved into the sample. The user rotates a latex rubber diskmounted on a snap flap to close the sample inlet 167 and places thecartridge into the analyzer.

(2) The analyzer makes contact with the cartridge, and a motor drivenplunger presses onto the foil pouch 161 forcing the wash/analysis fluidout into a central conduit 158.

(3) A separate motor driven plunger contacts the sample diaphragm 156pushing a measured segment of the sample along the sample conduit (fromreagent region R1 to R2). The sample is detected at the sensor chip 153via the conductivity sensors. The sensor chip is located in captureregion R3.

(4) The sample is oscillated by means of the sample diaphragm 156between R2 and R5 in a predetermined and controlled fashion for acontrolled time to promote binding to the sensor.

(5) The sample is pushed towards the waste region of the cartridge (R8)and comes in contact with a passive pump 157 in the form of a celluloseor similar absorbent wick. The action of wetting this wick seals thewick to air flow thus eliminating its ability to vent excess pressuregenerated by the sample diaphragm 156. The active vent becomes the“controlled air vent” of FIG. 16.

(6) Rapid evacuation of the sample conduit (effected by withdrawing themotor driven plunger from the sample diaphragm 156) forces a mixture ofair (from the vent) and wash/analysis fluid from the second conduit tomove into the inlet located between R5 and R4 in FIG. 16. By repeatingthe rapid evacuation of the sample conduit, a series of air separatedfluid segments are generated which are pulled across the sensor chiptowards the sample inlet (from R4 to R3 to R2 and R1). This washes thesensor free of excess reagents and wets the sensor with reagentsappropriate for the analysis. The wash/analysis fluid which originatesin the foil pouch can be further amended by addition of reagents in R7and R6 within the central wash/analysis fluid conduit.

(7) The wash/analysis fluid segment is drawn at a slower speed towardsthe sample inlet to yield a sensor chip which contains only a thin layerof the analysis fluid. The electrochemical analysis is performed at thispoint. The preferred method of analysis is amperometry but potentiometryor impedance detection is also used.

(8) And the mechanism retracts allowing the cartridge to be removed fromthe analyzer.

Example 5

In some embodiments, the device employs an immuno-reference sensor forpurposes of assessing the degree of non-specific binding occurringduring an assay. The immuno-reference sensor is fabricated in much thesame way as the analyte immunosensor with the exception that the immunoreagent is an anti-HSA (human serum albumin) antibody rather than ananti-analyte antibody. Upon exposure to a human whole blood or plasmasample, the reference sensor becomes coated with specifically bound HSA,an abundant endogenous protein present in all human blood samples thusaffording a common reference for all individual tests run using thepresent immunoassay format. NSB arising due to inadequate washing or dueto the presence of interferences can be monitored by means of thissecond sensor.

The net signal from the assay is comprised of the specific signalarising from the analyte immunosensor corrected by subtracting thenon-specific signal arising from the reference sensor, e.g., NetSignal=Analyte Sensor Signal−Reference Sensor Signal−Offset, as shown inequation 4 above. The “offset” is a coefficient that accounts for thedifference in the tendency of the two sensors to be subject to NSB. Ineffect, it accounts for the relative “stickiness” of each sensor withrespect to their ability to bind conjugate non-specifically and isestablished based on the responses of samples that are free of analyteand free of interference. This is done by independent experimentation.

The amount of signal tolerated at the reference sensor is subject tolimits defined by a quality control algorithm that seeks to safeguardthe integrity of results at low analyte concentration where the effectsof NSB have the greatest potential to affect assay results in a mannerthat can alter decision-making in a clinical environment. The essentialprincipal is that the existence of excessive signal at the referencesensor acts as a flag for the presence of NSB, due either to aninadequate wash step or interference.

Example 6

The experiments described below were performed on a BNP assay,specifically an i-STAT® BNP cartridge (Abbott Point of Care, Princeton,N.J., USA) or a partially modified version thereof.

In all of these embodiments, the use of one or more leukocidal reagentsand optionally beads opsonized for leukocytes, as described elsewhere inthis application may be integrated into the assay, for example thesample may be amended with both glucose oxidase, and with beadsopsonized for leukocytes, and optionally glucose and saponin. Generallythe sample is a whole blood sample, e.g., venous, arterial andcapillary, which may or may not have been amended with an anticoagulant.

As an experimental aid to understanding the affect of leukocytes onwhole-blood immunoassays, it is desirable to create an enriched buffy(blood) sample (EBS). The method adopted here was to take a spun tube ofblood, with plasma at the top, red cells at the bottom and a thin buffycoat in between. About half or more of the plasma and red cells werethen removed by pipette, and the sample remixed. In the experimentsdescribed herein, the EBS constituted approximately a ten-fold increasein the buffy concentration, i.e. roughly 90% of the plasma and red cellswere removed. The hematocrit value (Hct) of the sample was thereforemaintained in the normal range, roughly 50-60%.

FIG. 26 shows the effect of glucose oxidase on leukocyte activity. As abaseline indicator of leukocyte interference, a cut-off value of asignal rate of change above 0.05 nA per second, was used (see right sidey-axis, AmpActSlope). The data sets are broken into three groupsEBS+OX+Glu, EBS+OX and EBS+Glu, where OX is glucose oxidase at aconcentration of 4.0 IU per mL and Glu is glucose at a concentration of700 mg/dL. In the first group the enzyme is anticipated to removesubstantially all the oxygen and the co-reactant glucose is added insubstantial excess. The second group relies on glucose already in thesample to react with the enzyme. The third group is a control with addedglucose and no enzyme.

The left side of the y-axis in FIG. 26 shows both the glucose (mg/dL)and oxygen (PO₂ mm Hg) concentrations. The x-axis of FIG. 26 shows the“run start time,” which is the time between mixing the samples in eachgroup above, and taking an aliquot of that sample and injecting it intoa cartridge. Note that i-STAT® CG8+ cartridges (Abbott Point of Care,New Jersey, USA) were used to determine the glucose and oxygenconcentrations.

With respect to the first group (EBS+OX+Glu), the initial oxygenconcentration of about 200 mm Hg falls to about 10 mm Hg after 50minutes, and the glucose concentration drops from 700 to about 100 mg/dLover the same period. During this period immunoassays were run todetermine the AmpActSlope value. In this group 16 out of 19 assayedsamples had slopes below 0.05 nA per second. The AmpActSlope value isobtained from the analysis of current versus time signal traces, asshown in FIG. 25.

As shown in FIG. 25, the first trace beyond about the 50 second point isa steady current value, typical of a sample with a low or normalleukocyte activity towards the immunosensor. Specifically, theimmunosensor included a BNP immunosensor comprising a first captureantibody on bead attached to an electrode and a second signal antibodylabeled with ALP. A general description of this type of immunosensor andimmunoassay cartridge is found in U.S. Pat. No. 7,419,821 to Davis etal. The rate of change in the current is substantially less than thepreferred threshold value of 0.05 nA per second. The second trace istypical of a sample with a high leukocyte activity towards theimmunosensor, e.g., EBS. The rate of change in the current is greaterthan the preferred threshold value of 0.05 nA per second. The thirdtrace beyond about the 50 second point is a steady current value,typical of an EBS that has been treated with a combination of glucoseand glucose oxidase to reduce leukocyte activity towards theimmunosensor. Note that the rate of change in the current is once againsubstantially less than the preferred threshold value of 0.05 nA persecond.

With respect to the second group (EBS+OX), the oxygen concentration onlydrops down (from about 200 mm Hg) to about 50 mm Hg and the glucoseconcentration drops rapidly below about 20 mg/dL indicating that thereis insufficient glucose in the sample to consume all the oxygen. Thisobservation is related to the red blood cells acting as an oxygenbuffer, i.e., releasing more oxygen as the plasma concentration falls.During this period immunoassays were also run to determine theAmpActSlope value. In this group, 9 out of 13 assayed samples had slopesbelow 0.05 nA per second.

With respect to the third group (EBS+Glu), the oxygen concentration onlydrops slightly and the glucose concentration remains roughly unchanged.During this period immunoassays were run to determine the AmpActSlopevalue. In this group, 3 out of 8 assayed samples had slopes below 0.05nA per second.

Based on the data from the three groups, it is clear that glucoseoxidase can help contribute to a reduction in leukocyte activity andthat this is enhanced by the addition of excess glucose to the sample.It was found that a glucose oxidase concentration in excess of about 3IU per mL in the sample was preferable for venous samples and additionof glucose to give an initial sample concentration of above about 500mg/dL was preferable.

To further characterize these effects, a study was run on a largersample donor pool (15 donors multiple runs), as shown in FIG. 27. FIG.27 is a scatter plot where the desirable AmpActSlope value is less than0.05 and greater than −0.03 nA per second. In addition, in thisparticular experiment a maximum desirable sensor threshold current(ParamActDoc) was also set at 8.0 nA. The desirable result was thereforeto minimize samples falling into the five segments around the targetzone. Treated EBS+OX samples (squares) clearly show superior performanceto untreated samples (circles); treated N=287, 1.0% beyond threshold;untreated N=283, 13.4% beyond threshold; giving a 92.1% difference.Based on the experimental results, in general, the addition of theglucose oxidase reduces the number of assays where a slope above thedesirable threshold is observed by about 90%. This is a significantimprovement in overall assay quality and performance.

Those skilled in the art will recognize that while the present inventionis described in terms of a specific amperometric sensor used in animmunoassay, other parameters than AmpActSlope measured as delta nA persecond may be used as a determinant of leukocyte interference, e.g.,absolute current value, signal noise as fraction of signal strength andthe like. In addition, the present invention is equally relevant toother immunosensors, including electrochemical immunosensors based onpotentiometric and conductimetric measurements, and non-electrochemicalimmunosensors, e.g., surface acoustic wave, surface plasmon resonance,optical waveguides and the like. Each of these has known criteria forassessing adverse effects on sensor performance. These can be assessedfor suitability using the EBS method, or variants thereof

FIG. 28A shows an additional study that was performed to furthercharacterize the use of EBS+OX+Glu treated samples. The “Sample 2” setof data was obtained where the treatment occurs in a closed syringe toprevent any oxygen being introduced from ambient air. Over ten minutesthe oxygen dropped from about 200 mm Hg to about 20 mm Hg and theglucose dropped from about 600 to about 360 mg/dL. All 12 consecutiveimmunoassays run on the sample had desirable AmpActSlope values below0.05 nA per second. The “Sample 3” data reflects opening the syringeabout 20 minutes after the initial runs to allow ambient air access.This sample was run at the 40 minute mark. Here, the leukocytes are nolonger deprived of oxygen and an increased number of immunoassay testsshow AmpActSlope values above the desired threshold. The “Sample 1” datain FIG. 28A is a control EBS+Glu only. FIG. 28B presents the data in asimpler form with the points at run times of 1 and 10 minutes beingthose of the syringe samples and those at 40 minutes being the samesample after re-oxygenation by exposure to air.

While it is more common for a blood sample used in an immunoassayperformed in a laboratory to be drawn from a vein, where point-of-care(or bedside, e.g. Emergency Room, Operating Room) testing is done, it isnot uncommon for the sample to be drawn from an artery. Obviously thePO₂ concentration will generally be higher in the latter type of sample.To assess the impact of an arterial sample on the present assay,simulated arterial samples were generated and tested. Those skilled inthe art will recognize that directly obtaining arterial samples involvesa much higher risk of internal bleeding, hence the use of simulatedsamples. Simulated samples were created with a tonometer using venousblood collected in an EDTA tube. The tonometer equilibrates the bloodwith a predetermined gas composition.

FIG. 29 shows data for the simulated arterial samples. Note thesesamples were not enriched as described above. The first column of data(WB) indicates that the tonometered whole blood sample had an initialPO₂ of about 150 mm Hg and an initial glucose value of about 70 mg/dL,and that neither changed significantly over a period of eight minutes.Addition of glucose oxidase at both 6 and 12 IU per mL (see WB+6 andWB+12 columns) resulted in both decreased glucose and PO₂. Asanticipated in the fourth column WB+Glu, the glucose was elevated andthe PO₂ remained substantially unchanged. Both the fifth and sixthcolumns (WB+6+Glu and WB+12+Glu) showed drops in PO₂ and glucose. Asillustrated in FIG. 29, adding glucose oxidase at 6.0 IU and 12.0 IU permL (and preferably in excess of about 3.0 IU per mL), is sufficient toinhibit phagocytosis in arterial blood through its ability to reduce thepartial pressure of oxygen in the sample below about 50 mm Hg.

Example 7

As described herein, the use of saponin as a leukocidal reagent is analternative method for inhibiting the activity of leukocytes. FIG. 30shows the effect of added saponin (x-axis values in mg/mL in the range 0to 10 mg/mL) against the analysis slope with the threshold value of 0.05nA per second indicated for EBS samples. This study shows that sampleswith 0.5 mg/mL of saponin exhibit significantly fewer slopes above thethreshold than untreated samples (Prod). FIG. 30 also shows similarresults at concentrations up to 10 mg/mL. In other experiments it wasfound that saponin concentrations in the range of about 0.1 to 20 mg/mLwere useful in reducing leukocyte activity. It was also found thatsaponin may have an effect on the precision of the assay, that wasameliorated by repeated wash cycles. Consequently, in certainapplications lower but effective saponin concentrations around 0.5 to1.0 mg/mL may be preferred.

While the present invention as described above is generally directed toreducing or eliminating interference from leukocytes in an analyteimmunoassay with a whole blood sample, it is also applicable toimmunoassays performed in other types of biological samples whereleukocytes may be present, e.g., cerebrospinal fluid. In addition it isapplicable to samples where residual leukocytes may be present despitean intention to remove them by centrifugation or filtration, e.g.,plasma. It is also applicable to samples that may have been diluted,e.g., with a buffer. Furthermore, while the invention is generallydirected to amending the sample by dissolving into the sample a dryreagent, it is also practical in other embodiments to add the reagent,e.g., the one or more leukocidal reagents, as a liquid to the sampleduring the analysis or during sample collection. It is also apparentthat the present invention has been described herein in terms ofelectrochemical detection approaches, e.g., amperometric andpotentiometric approaches, although it is equally applicable to otherdetection modes, notably optical sensing approaches such asluminescence, fluorescence and absorbance based approaches.

While the invention has been described in terms of various preferredembodiments, those skilled in the art will recognize that variousmodifications, substitutions, omissions and changes can be made withoutdeparting from the spirit of the present invention. For example, whileportions of the description are directed to non-competitive sandwichimmunoassays, the devices and processes of the invention similarly maybe employed in competitive immunoassays. Additionally, it is intendedthat the scope of the present invention be limited solely by the scopeof the following claims.

What is claimed is:
 1. A kit for performing an electrochemicalimmunoassay for a target analyte in a whole blood sample comprising: aleukocidal reagent configured to substantially inhibit phagocyticactivity of leukocytes in the whole blood sample, wherein the leukocidalreagent is a mitochondrial electron transport inhibitor or amitochondrial membrane uncoupling agent, and electrochemical immunoassayreagents for detecting at an immunosensor the target analyte in theleukocidal reagent-treated whole blood sample.
 2. The kit of claim 1,wherein the leukocidal reagent is the mitochondrial electron transportinhibitor.
 3. The kit of claim 1, wherein the leukocidal reagent is themitochondrial membrane uncoupling agent.
 4. The kit of claim 1, whereinthe leukocidal reagent is the mitochondrial electron transport inhibitoror the mitochondrial membrane uncoupling agent and selected from thegroup consisting of cyanide, antimycin, rotenone, malonate, carbonylcyanide p-[trifluoromethoxyl]-phenyl-hydrozone, 2,4-dinitrophenol, andoligomycin.
 5. The kit of claim 1, further comprising the immunosensorcomprising an immobilized first antibody to the target analyte, and alabeled second antibody to the target analyte, wherein the immunoassayis a sandwich assay.
 6. The kit of claim 1, further comprising theimmunosensor comprising an immobilized antibody to the target analyte,and the target analyte is labeled, wherein the immunoassay is acompetitive assay.
 7. The kit of claim 1, further comprising beadsopsonized for the leukocytes.
 8. The kit of claim 1, wherein the wholeblood sample is amended with an anticoagulant.
 9. The kit of claim 1,wherein the target analyte is selected from the group consisting ofTroponin I (TnI), Troponin T (TnT), Brain Natriuretic Peptide (BNP),N-terminal of the Prohormone Brain Natriuretic Peptide (NTproBNP),Prohormone Brain Natriuretic Peptide (proBNP), Human ChorionicGonadotropin (HCG), Thyroid-Stimulating Hormone (TSH), NeutrophilGelatinase Associated Lipocalin (NGAL), theophylline, digoxin, andphenytoin.
 10. The kit of claim 1, further comprising the immunosensor.11. The kit of claim 10, wherein the leukocidal reagent is positionedupstream of the immunosensor such that the whole blood sample iscontacted with the leukocidal reagent prior to contacting theimmunosensor.